Citation
Molecular cloning and characterization of Bacteroides gingivalis antigens

Material Information

Title:
Molecular cloning and characterization of Bacteroides gingivalis antigens
Creator:
Tumwasorn, Somying, 1953-
Publication Date:
Language:
English
Physical Description:
ix, 118 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Antigens ( jstor )
Antiserum ( jstor )
Bacteria ( jstor )
Bacteroides ( jstor )
DNA ( jstor )
Gels ( jstor )
Hemagglutination ( jstor )
Plasmids ( jstor )
Rabbits ( jstor )
Bacteroides -- pathogenicity ( mesh )
Cloning, Molecular ( mesh )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( mesh )
Escherichia Coli ( mesh )
Gingival Diseases -- etiology ( mesh )
Immunology and Medical Microbiology thesis Ph.D ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1988.
Bibliography:
Bibliography: leaves 108-117.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Somying Tumwasorn.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030455361 ( ALEPH )
20338193 ( OCLC )
AET3588 ( NOTIS )

Downloads

This item has the following downloads:

ER8XORGYJ_2IUIY5.xml

molecularcloning00tumw.pdf

molecularcloning00tumw_0018.txt

molecularcloning00tumw_0016.txt

molecularcloning00tumw_0045.txt

molecularcloning00tumw_0009.txt

molecularcloning00tumw_0066.txt

ER8XORGYJ_2IUIY5_xml.txt

molecularcloning00tumw_0035.txt

molecularcloning00tumw_0077.txt

molecularcloning00tumw_0092.txt

molecularcloning00tumw_0128.txt

molecularcloning00tumw_0116.txt

molecularcloning00tumw_0068.txt

molecularcloning00tumw_0103.txt

molecularcloning00tumw_0040.txt

molecularcloning00tumw_0087.txt

molecularcloning00tumw_0119.txt

molecularcloning00tumw_0036.txt

molecularcloning00tumw_0015.txt

molecularcloning00tumw_0069.txt

molecularcloning00tumw_0084.txt

molecularcloning00tumw_0117.txt

AA00009090_00001_pdf.txt

molecularcloning00tumw_0029.txt

molecularcloning00tumw_0024.txt

molecularcloning00tumw_0091.txt

molecularcloning00tumw_0048.txt

molecularcloning00tumw_0012.txt

molecularcloning00tumw_0030.txt

molecularcloning00tumw_0053.txt

molecularcloning00tumw_0010.txt

molecularcloning00tumw_0098.txt

molecularcloning00tumw_0120.txt

molecularcloning00tumw_0028.txt

molecularcloning00tumw_0054.txt

molecularcloning00tumw_pdf.txt

molecularcloning00tumw_0083.txt

molecularcloning00tumw_0082.txt

molecularcloning00tumw_0125.txt

molecularcloning00tumw_0110.txt

molecularcloning00tumw_0051.txt

molecularcloning00tumw_0034.txt

molecularcloning00tumw_0005.txt

molecularcloning00tumw_0100.txt

molecularcloning00tumw_0090.txt

molecularcloning00tumw_0088.txt

molecularcloning00tumw_0023.txt

molecularcloning00tumw_0037.txt

molecularcloning00tumw_0000.txt

molecularcloning00tumw_0065.txt

molecularcloning00tumw_0019.txt

molecularcloning00tumw_0064.txt

molecularcloning00tumw_0123.txt

molecularcloning00tumw_0105.txt

molecularcloning00tumw_0047.txt

molecularcloning00tumw_0122.txt

molecularcloning00tumw_0071.txt

molecularcloning00tumw_0026.txt

molecularcloning00tumw_0008.txt

AA00009090_00001.pdf

molecularcloning00tumw_0080.txt

molecularcloning00tumw_0086.txt

molecularcloning00tumw_0060.txt

molecularcloning00tumw_0067.txt

molecularcloning00tumw_0049.txt

molecularcloning00tumw_0002.txt

molecularcloning00tumw_0004.txt

molecularcloning00tumw_0062.txt

molecularcloning00tumw_0126.txt

molecularcloning00tumw_0099.txt

molecularcloning00tumw_0061.txt

molecularcloning00tumw_0070.txt

molecularcloning00tumw_0106.txt

molecularcloning00tumw_0044.txt

molecularcloning00tumw_0078.txt

molecularcloning00tumw_0052.txt

molecularcloning00tumw_0072.txt

molecularcloning00tumw_0050.txt

molecularcloning00tumw_0055.txt

molecularcloning00tumw_0104.txt

molecularcloning00tumw_0043.txt

molecularcloning00tumw_0130.txt

molecularcloning00tumw_0039.txt

molecularcloning00tumw_0121.txt

molecularcloning00tumw_0075.txt

molecularcloning00tumw_0118.txt

molecularcloning00tumw_0108.txt

molecularcloning00tumw_0001.txt

molecularcloning00tumw_0073.txt

molecularcloning00tumw_0056.txt

molecularcloning00tumw_0011.txt

molecularcloning00tumw_0113.txt

molecularcloning00tumw_0033.txt

molecularcloning00tumw_0022.txt

molecularcloning00tumw_0115.txt

molecularcloning00tumw_0013.txt

molecularcloning00tumw_0129.txt

molecularcloning00tumw_0021.txt

molecularcloning00tumw_0057.txt

molecularcloning00tumw_0027.txt

molecularcloning00tumw_0014.txt

molecularcloning00tumw_0111.txt

molecularcloning00tumw_0081.txt

molecularcloning00tumw_0095.txt

molecularcloning00tumw_0102.txt

molecularcloning00tumw_0089.txt

molecularcloning00tumw_0076.txt

molecularcloning00tumw_0017.txt

molecularcloning00tumw_0112.txt

molecularcloning00tumw_0093.txt

molecularcloning00tumw_0059.txt

molecularcloning00tumw_0025.txt

molecularcloning00tumw_0038.txt

molecularcloning00tumw_0085.txt

molecularcloning00tumw_0114.txt

molecularcloning00tumw_0107.txt

molecularcloning00tumw_0032.txt

molecularcloning00tumw_0074.txt

molecularcloning00tumw_0109.txt

molecularcloning00tumw_0097.txt

molecularcloning00tumw_0020.txt

molecularcloning00tumw_0041.txt

molecularcloning00tumw_0124.txt

molecularcloning00tumw_0042.txt

molecularcloning00tumw_0031.txt

molecularcloning00tumw_0063.txt

molecularcloning00tumw_0094.txt

molecularcloning00tumw_0006.txt

molecularcloning00tumw_0101.txt

molecularcloning00tumw_0007.txt

molecularcloning00tumw_0058.txt

molecularcloning00tumw_0046.txt

molecularcloning00tumw_0003.txt

molecularcloning00tumw_0127.txt

molecularcloning00tumw_0079.txt

molecularcloning00tumw_0096.txt


Full Text












MOLECULAR CLONING AND Ci~\-RACTERIZATION OF
PA.CTEROIDES GINGIVALIS A\XTIGE'.S




















BY

SOMYING TUMWASORN


A DISSERTATION PRESENTED TO TIHE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1988




MOLECULAR CLONING AND CHARACTERIZATION OF
PACTEROIDES GINGIVALIS ANTIGENS
BY
SOMYING TUMWASORN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOO
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988


ACKNOWLEDGEMENTS
I would like to express my appreciation to my advisor, Dr. Ann
Progulske for her guidance, friendship, and assistance in this study
and in the preparation of this dissertation. I have enjoyed working
with her and realize that I truly like tills kind of research.
I really appreciate Dr. Donna H. Duckworth, my committee member,
for her guidance and technical assistance in the area of molecular
genetics and for her moral support and friendship since the beginning
of my graduate study.
Special thanks go to my committee members, Dr. Clay B. Walker, Dr.
Wlliam B. Clark, Dr. William P. McArthur, Dr. Anthony F. Barbet, my
external examiner, Dr. Francis L. Macrina, and the chairman of the
Department of Immunology and Medical Microbiology, Dr. Richard R.
Moyer, for their constructive criticisms and technical assistance in
this study.
I also thank the Fulbright Foundation for providing financial
support for the beginning of my study and the Thai Government for
granting me a leave of absence.
I would also like to thank the fellow' graduate students and
scientists, Dr. Connie D. Young, and Dr. Thomas A. Brown for their
technical assistance, discussions and friendship.
I wish to express my deepest gratitude to my parents Sanguan and
Chaufa Juijaitrong who provided me wdth the educational background and


encouragement, their love, concern, and devotion.
Finally, I wish to extend my appreciation to my husband Sornthep
and sons, Pattarawuth and Nattapol, for their love, patience,
encouragement and dedication that created the necessary environment to
permit the conclusion of this work.


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT viii
CHAPTER
ONE INTRODUCTION 1
Bacteroides gingivalis as the Periodontopathogen 1
Pathogenicity of B. gingivalis 2
Application of Recombinant DNA Techniques to
the Study of Periodontal Disease 9
TWO CLONING AND EXPRESSION OF BACTEROIDES GINGIVALIS
ANTIGENS IN ESCHERICHIA COLI 12
Introduction 12
Materials and Methods 14
Results 24
Discussion 42
THREE CHARACTERIZATION OF BACTEROIDES GINGIVALIS ANTIGENS
SYNTHESIZED IN ESCHERICHIA COLI 48
Introduction 48
Materials and Methods 50
Results 55
Discussion 97
FOUR CONCLUSION 103
LITERATURE CITED 108
BIOGRAPHICAL SKETCH 118
iv


LIST OF TABLES
Table Page
1 Characterization of E. coli transformants which
express B. gingival is antigens 26
2 Titer of anti-£b gingivalis against E. coli
transformants which express B. gingivalis antigens 39
3 Inhibition of adherence to SHA by adsorbed anti-fi.
gingivalis antisera 56
4 Inhibition of hernagglutinating activity of B.
gingivalis by anti-hemagglutinating E. coli
antisera 96
5 Inhibition of hernagglutinating activity of B.
gingivalis by adsorbed anti-fb gingivalis
antiserum 98
v


LIST OF FIGURES
Figure Page
1 Map of pUC 9 16
2 Agarose gel electrophoresis of recombinant plasmids 28
3 Agarose gel electrophoresis of different restriction
digests of recombinant plasmid from clone 3 31
4 Agarose gel electrophoresis of different restriction
digests of recombinant plasmids from clones 5, 6, 7,
and 8 33
5 Agarose gel electrophoresis of different restriction
digests of recombinant plasmids from clones 1, 2,
and 4 35
6 Hybridization of recombinant plasmids with 3ZP labeled
B. gingivalis DNA probe 38
7 SDS-PAGE (on 12.5% acrylamide) and Western blot analysis
of expressed B. gingivalis antigens 41
8 SDS-PAGE (on 5% acrylamide) of expressed B. gingivalis
antigen in clone 2 44
9 Hemagglutination of sheep erythrocytes 59
10 Agarose gel electrophoresis of restriction digests of
the recombinant plasmid from clone 2 62
11 Agarose gel electrophoresis of restriction digests of
the recombinant plssmid from clone 5 64
12 Agarose gel electrophoresis of restriction digests of
recombinant plasmids from clones 5 and 7 66
13 Restriction map of the recombinant plasmid from
clone 2 68
14 Restriction map of the recombinant plasmid from
clone 5 70
15 Restriction map of the recombinant plasmid from
clone 7 72
16 Schematic diagram of restriction enzyme recognition
sites of recombinant plasmids from clones 2, 5,
and 7 74
17 Southern blot analysis of the hemagglutinating
E. coli 76
Vi


Figure Page
18 Agarose gel electrophoresis of recombinant plasmids
from clones 5.1, 5.2, 5.3, and 5.4 80
19 Western blot analysis of native B. gingivalis
antigens expressed by clone 2 83
20 Western blot analysis of native B. gingivalis
antigens expresses by clone 2 85
21 Western blot analysis of native B. gingivalis
antigen expressed by clone 7 88
22 Western blot analysis of native B. gingivalis
antigens expressed by clones 2, 5, and 7 90
23 Detection of B. gingivalis antigens synthesized
by clones 2, 5, arid 7 as determined by Western blot
analysis 93
24 ELISA of anti-clone 2 antiserum adsorbed with
various numbers of cells 95


Abstract of a Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MOLECULAR CLONING AND CHARACTERIZATION OF
BACTEROWES GINGIVALIS ANTIGENS
By
Somying Tumwasorn
April 1988
Chairman: Ann Progulske
Major Department: Immunology and Medical Microbiology
Bacteroides gingivalis, a Gram-negative anaerobic bacterium, is
strongly implicated as an etiological agent of periodontal disease.
However, its exact role in the disease process has not yet been
established. Recombinant DNA technology w-as applied as an initial
approach to a molecular study of B. gingivalis antigens by preparing
genomic libraries of B. gingivalis strain 381 in E. coli JM 109 via a
pUC 9 plasmid vector. Detection of the expression of B. gingivalis
antigens wras achieved by using E. coli adsorbed rabbit anti-5.
gingivalis sera.
Five different clones w-ere found to stably exhibit B. gingivalis
antigen expression. Characterization of the antigen-expressing clones
demonstrated that clones 2, 5, and 7 agglutinate sheep erythrocytes
whereas E. coli JM 109 (pUC 9) does not. Clones 5 and 7 w'ere found to
have one insert fragment in common and this insert was found to have
little or no homology to the insert of clone 2. Clone 5 is also able
vi i i


to autoagglutinate and it was found that a 760 bp DNA fragment codes
for this activity. The common insert of clones 5 and 7 appears to
have a Bacteroides promoter and to code for the hemagglutinating
activity of these clones. The clone 2 insert does not have a
Bacteroides promoter and is under the control of plasmid lac promoter.
Antisera against clones 2, 5, and 7 were found to inhibit the
hemagglutinating activity of B. gingivalis whereas adsorption of
anti-5, gingivalis antiserum with clones 2, 5, and 7 partially removed
the hemagglutination inhibition activity. However, these clones do not
remove the saliva-treated hydroxyapatite (SHA) adherence inhibition
activity of anti-5, gingivalis antiserum. Western blot analysis of
5. gingivalis cell lysate antigens using E. coli adsorbed antisera
against clones 2, 5, and 7 demonstrated that all antisera reacted to 2
major bands of MWs 43,000 and 38,000, which have been reported to be
the major bands of the 5. gingivalis hemagglutinin. E. coli
adsorbed anti-clones 5 and 7 antisera did not react to the 125,000
protein band expressed in clone 2. In addition, adsorption assays
demonstrated that the epitope of the expressed antigen in clone 2 is
not related to that of clones 5 and 7. A number of experiments are
proposed to further characterize the 5. gingivalis hemagglutinin genes,
the nat:ve hemagglutinin molecules, and the significance of
hemagglutinins in periodontal disease.
i x


CHAPTER ONE
INTRODUCTION
Periodontal disease (PD) is a chronic inflammatory disease which
results in the destruction of the supporting tissues of teeth (Kagan,
1980). Although the specific microbial etiology of PD is not known, it
is widely accepted that bacteria are the contributing agents of the
disease for the following reasons (Socransky, 1977): 1) disease
correlates with the presence of plaque, 2) antibiotics are effective in
treatment of PD, and 3) implantation of certain genera of bacteria into
gnotobiotic rats results in PD of infected but not of control rats.
Bacteroides gingivalis as the Periodontopathogen
The presence of a complex microflora in the subgingival crevice
has complicated the identification of the specific etiologic agents of
PI). However, several studies (Socransky, 1977; Slots, 1979; White ai d
Mayrand, 1981) indicate that a few genera, primarily Gram-negative
anaerobes, appear to be associated with disease progression. For
example, the proportion of Gram-negative anaerobes, especially black-
pigmented Bacteroides, increases markedly in the subgingival flora with
increasing severity of PD. Bacteroides gingivalis, previously oral
Bacteroides asaccharolyticus (Coykendall et al., 1980), is the black-
pigmented Bacteroides which has emerged as a key putative
periodontopathogen for a number of compelling reasons.
1


2
B. gingivalis is the predominant bacterial species isolated from
periodontal lesions of patients with severe adult periodontitis
(Slots,1977; Tanner et al., 1977). Patients with adult periodontitis
have been found to have higher levels of IgG antibodies to B. gingivalis
than normal adults (Mouton et. al., 1981) and local immunity to B.
gingivalis is greater in the more advanced cases than in the early
forms of PD (Kagan, 1980). Serum antibody titers to B. gingivalis have
been reported to decrease after therapy of adult periodontitis
patients, suggesting that antibodies to B. gingivalis result from
infection of this organism (Tolo et al., 1982). B. gingivalis is also
the most interesting and potentially virulent bacterium cultivable from
the subgingival crevice with respect to its capacity for breakdown of
tissues and host defense mechanisms (Mayrand and McBride, 1980; Van
Steenbergen et al., 1982; Nilsson et al., 1985). In addition, B.
gingivalis appears to be a causative agent of experimental
periodontitis in animals. When B. gingivalis is implanted as the
monocontaminant in gnotobiotic rats, it causes accelerated alveolar
bone loss (CrawTord et al., 1977). In a longitudinal study of alveolar
bone loss in Macaca arctoides (Slots and Hausmann, 1979), the proportion
of B. gingivalis-type isolates reportedly increased from a minority of
the cultivable microbiota prior to bone loss to a majority of the
microflora when alveolar bone loss was detectable.
Pathogenicity of B. gingivalis
Although B. gingivalis has been strongly implicated as an
etiological agent of adult periodontitis, its exact role in the disease
process has not yet been established. In order to produce PD, it is


3
likely that bacteria and/or their products may lead to the destruction
of the gingival tissues by direct action or indirectly by eliciting an
immune response which is detrimental to the host tissues.
Periodontopathic bacteria such as B. gingivalis must possess
characteristics which enable them to colonize the host, survive in the
periodontal pocket, possibly invade the gingival tissues, and to
destroy the collagenous periodontal ligament, the alveolar bone, and
other tissue components surrounding the tooth (Slots and Genco, 1984).
Colonization
It is now recognized that colonization of the oral cavity and many
other mucosal environments requires the adherence of bacteria to the
surface in order to resist the cleansing action of glandular secretions
(Gibbons and Van Houte, 1975; 1980). The adherence of bacteria to host
tissues is thus a prerequisite for colonization, which is the initial
event in the pathogenesis of disease (Gibbons and Van Houte, 1975).
The mechanisms of bacterial adherence involve both ionic and other
physical (covalent) forces. Many, if not all pathogenic bacteria
possess specific ligands on their surfaces, called "adhesins" which
bind to complementary components on host tissues (Gibbons and Van
Houte, 1980). The mechanisms of adherence may involve the interaction
of carbohydrate binding proteins, or lectins, on bacterial surfaces
with carbohydrate-containing receptors on host cells. Binding
properties of adhesins may also be facilitated by their hydrophobic
domains (Gibbons, 1984).


4
Components of bacteria which mediate attachment to host tissues
include surface structures such as fimbriae, capsular materials,
lipopolysaccharides, and membrane-associated extracellular vesicles
(Slots and Genco, 1984). In the oral cavity, bacteria can attach to
host tissues as well as Gram-positive bacteria in pre-formed plaque
(Slots and Gibbons, 1978). The nature of the binding sites on teeth
and oral tissues to which Gram-negative bacteria attach has not been
well established. In vitro, B. gingivalis can attach to and agglutinate
erythrocytes (Okuda and Takazoe, 1974; Slots and Gibbons, r. 78; Slots
and Genco, 1979; Okuda et al., 1981), can adhere in high numbers to
human buccal epithelial cells (Slots and Gibbons, 1978; Okuda et al.,
1981), crevicular epithelial cells derived from periodontal pockets
(Slots and Gibbons, 1978), and surfaces of Gram positive bacteria
present, in plaque, (Slots and Gibbons, 1978; Schwarz et al., 1987). B.
gingivalis is also able to adhere to untreated and saliva-treated
hydroxyapatite, but in comparatively low numbers (Slots and Gibbons,
1978). B. gingivalis has also been reported to bind to HR9 matrix, a
material similar to the basement membrane barrier underlying connective
tissue (Leong et al., 1985). Recently, it has been reported that B.
gingivalis can bind fibrinogen and possibly colonize host tissues by
attaching to fibrinogen-coated surfaces (Lantz et al., 1986).
Bacterial antagonism may also play an important role in mediating
the colonization of B. gingivalis. In normal adults, Streptococcus
sanguis is a predominant organism in supra- and subgingival plaque.
S. sanguis elaborates sanguicin, a bacteriocin which in vitro inhibits
black-pigmented Bacteriodes (Nakamura et al., 1981). Experimental
studies in humans have shown that the number of Streptococcus species,


5
including >9. sanguis, are decreased, while those of Actinomyces and
black pigmented Bacteriodes are increased (Leosche and Syed, 1978) in
gingivitis. The mechanism of the proportional decrease of S. sanguis
is not. known but this shift seems to be one of the triggers for the
initiation of compositional changes in the subgingival flora. A
decrease of sanguicin production may permit the growth of Actinomyces
species and black pigmented Bacteriodes (Takazoe et al., 1984). The
growth of B. gingivaiis may be enhanced by herriiri when bleeding occurs
in gingivitis, since hemiri is a required factor for the cultivation of
B. gingivaiis. Recently, it has been reported that B. gingivaiis grown
under hemin-limited conditions has a reduced virulence in mice compared
with bacteria cultured in an excess of hemin (McKee et al., 1986).
When colonization of B. gingivaiis occurs, there seems to be a
change in the bacterial composition in the periodontal pocket. This
could be explained by studies of Nakamura et al (1978; 1980) which have
demonstrated that B. gingivaiis produces the black pigment hematin
which inhibits the growth of some Gram-positive bacteria, including S.
mutans, S. mitis, A. viscosus, A. naeslundii, A. israelii, Bacterionema
matruchotii, Corynebacterium parvum arid Propionibacterium acns.
Factors other than inhibitory substances could also affect the
colonization of B. gingivaiis, i.e., the nature of specific antibody
and other components in gingival fluid as well as the interactions
between the new predominant colonizer and other pre-existing
residents (Takazoe et al., 1984).


6
Evasion of Host Defense
B. gingivalis may survive in the periodontal pocket because it
resists phagocytosis. Sundqvist et al. (1982) demonstrated that in
vitro, most strains of B. gingivalis exhibit a higher resistance to
phagocytosis than do less pathogenic strains and that impaired
phagocytosis of this bacterial species is related to capsular material.
The Bacteroides capsule only poorly activates complement, therefore it
may function to decrease PMN chemotactic stimulus by masking LPS which
strongly activates complement (Okuda et al., 1978). Various
experiments have verified that the black pigmented Bacteroides strains
do not stimulate a strong PMN chemotactic response (Sveen, 1977 a,b;
Lindhe and Socransky, 1979; Sundqvist and Johansson, 1980).
Most strains of B. gingivalis demonstrate resistance to serum
bactericidal systems (Sundqvist and Johansson, 1982). B. gingivalis
has also been shown to degrade the plasma proteins which are important
in the host defense, such as the complement factors C3 and C5
(Sundqvist et al., 1985), immunoglobulins G, A, and M (Kilian, 1981;
Sundqvist et al., 1985), alpha-l-proteinase inhibitor,
alpha-2-macroglobuiin (Carlsson et al., 1984a), haptoglobin, and
hemopexin (Carlsson et al., 1984b). It has also been shown that
B. gingivalis has the capacity to inactivate and degrade the plasma
proteins of importance in the initiation and control of the
inflammatory response such as Cl- inhibitor, antithrombin, and aipha-2-
antiplasmin (Nilsson et al., 1985). In addition, B. gingivalis can
degrade fibrinogen (Lantz et al., 1986) and fibrin (Mayrand and
McBride, 1980; Wikstrom et al., 1983); therefore, no effective fibrin
barrier is formed around the organism. B. gingivalis thus appears to


7
be an organism fully capable of inactivating the host defense
mechanisms against invading bacteria.
Periodontal Tissue Destruction
B. gingivalis possesses a number of components with the potential
to destroy gingival tissue constituents as follows: The B. gingivalis
lipopolysaccharide possesses strong bone resorptive activity (Nair et
al., 1982), and inhibits the growth of cultured fibroblasts derived
from healthy and periodontally diseased human gingiva (Layman and
Diedrich, 1987). The lipopolysaccharide is also a suspected component
that stimulates mononuclear cells to produce a factor which strongly
stimulates osteoclast-mediated mineral resorption (Bom-Van Noorloos et
al., 1986). B. gingivalis proteolytic enzymes, especially collagenase
(Mayrand and McBride, 1980; Robertson et al., 1982; Mayrand and
Grenier, 1985) and a trypsin-like protease (Slots, 1981; Laughon et
al., 1982) may be directly involved in periodontal tissue destruction.
Enzymes other than proteases may also play an important role in the
pathogenesis of periodontal disease. For example, alkaline and acid
phosphatases (Slots, 1981; Laughon et al., 1982) may cause alveolar
bone breakdown since it has been shown that bacterial phosphatases
could cause alveolar bone breakdown (Frank and Voegel, 1978).
Bacterial products, i.e., butyrate, propionate (Singer and Buckner,
1981) and volatile sulfur compounds (Tonzetich and McBride, 1981) are
also suspected to be toxic to periodontal tissues. Recently, it has
been reported that B. gingivalis possesses a cartilage-degrading ability
which is suspected to be due to its ability to degrade proteinase
inhibitors (Klamfeldt, 1986).


8
It has been suggested that periodontal tissue destruction is
mediated not only by bacteria and their products but aiso by the host
defense mechanisms (Horton et ah, 1974; Nisengard, 1977). For
example, cell mediated immunity has been shown to correlate with the
periodontal status of patients (Ivanyi and Lehner, 1970; Patters et
al., 1976; Patters et al., 1979). B. gingivalis was found to stimulate
significantly more lymphoproliferative response iri patients with
destructive periodontitis than those of normal subjects or those with
gingivitis (Patters et al., 1980). A lymphoproliferative response
results in the production of lymphokines, several of which can account
for some of the destructive effects in periodontal disease. For
example, alpha-lymphotoxin can cause cell death and osteoclast
activating factor can stimulate osteoclastic bone resorption (Horton et
al., 1972). Macrophages have also been suggested to play a role in
periodontal tissue destruction. Macrophages may be stimulated by
bacterial antigens such as LPS (Wahl, 1982), or by lymphokines (Mooney
and Waksman, 1970; Wahl et al., 1975), and subsequently produce
tissue-degrading enzymes such as collagenase and other proteases (Wahl
et al., 1975; Wahl, 1982). Recently, it has been demonstrated that
lipopolysaccharide of B. gingivalis can induce circulating mononuclear
cells to release collagenase-inducing cytokines. The cytokines then
induce collagenase synthesis in human gingival fibroblasts (Health et
al., 1987). In addition, IgE, mast cells and basophils may also play a
role in periodontal disease (Jayawardene and Goldner, 1977; Olsson-
Wennstrom et al., 1978).


9
Application of Recombinant DNA
Techniques to the Study of Periodontal Disease
The recombinant DNA techniques developed during the past few years
have proven t be powerful tools for the study of pathogenesis.
Several major antigens and virulence factors have been cloned as a
means of further characterizing their chemical natures, genetic
regulation, and function in various diseases. For example, the cloning
and expression of the Neisseria gonorrhoeae pilus protein in E.
coli (Meyer and So, 1982) has helped explain the molecular mechanism of
antigenic variation. In other studies, the cloning of several
virulence factors including exotoxins (Vodkin and Leppla, 1983; Vasil
et al., 1986; Nicosia et al., 1987), enterotoxiris (Pearson and
Mekalanos, 1982), a hemolysin (Goldberg and Murphy, 1984), and a
pneumolysin (Walker et al., 1987) have allowed genetic studies of these
proteins and have facilitated the production of safer vaccines.
Cloning antigens encoded by unknown genes is made possible by preparing
a genomic library in which any gene is theoretically represented. If
the number of clones is large enough, it is hoped that any gene can be
isolated by screening the library (Perbal, 1984). Genomic libraries of
both Treponema pallidum (Stamm et al., 1982) and Legionella pneumophila
(Engleberg et al., 1984 a;b) have been made as a first step in
isolating and characterizing their major surface antigens.
The recombinant DNA techniques have, however, been applied only
sparingly to the study of Gram-negative anaerobic pathogens and even
less to the study of the molecular mechanisms of periodontopathogenesis.
The recombinant DNA methodologies offer advantages over previous
methods used in the study of oral pathogens. Since several potential


10
periodontopathogens, including B. gingivaJis, are difficult to grow to
high densities, isolation and purification of antigens, especially
those present in small amounts, are often difficult and tedious because
of a limited amount of starting material. Cloning specific structures
in an organism such as E. coli would greatly alleviate these problems
since E. coli can be grown to high densities easily and cloned structures
can be overproduced in E. coli (De Franco et al., 1981; Matsumura et al.,
1986). This would facilitate the isolation and purification of that
structure or component. Also, the cloning and expression of antigens
would isolate the antigens at the genetic level. The cloned antigens
can then be prepared as products devoid of other B. gingivalis antigens.
Thirdly, the cloning of B. gingivalis antigens would allow a genetic and
molecular analysis of the gene(s) which is presently difficult to do
due to the lack of a genetic system in B. gingivalis. Cloning antigens
which may be protective or have potential virulence properties is an,
as yet, relatively unexplored approach to define the role of B.
gingivaJis in periodontal disease. It is an approach that may lead to
a more complete understanding of the molecular mechanisms of
periodontal disease as well as providing molecular tools for the future
production of a vaccine for periodontal disease.
The purpose of this study was to employ recombinant DNA techniques
to clone antigens of B. gingivalis as an initial step in defining their
roles in pathogenesis. The specific aims were to
1. Construct genomic libraries (clone banks) of B. gingivalis
chromosomal DNA in E. coli.
2. Identify E. coli. transformants which express B. gingivalis
antigens.


11
3. Identify cloned antigens which are potential virulence factors.


CHAPTER TWO
CLONING AND EXPRESSION OF BACTEROWES GINGIVALIS
ANTIGENS IN ESCHERICHIA COLI
Introduction
Several lines of evidence strongly implicate Bacteroides
gingivalis, a Gram-negative anaerobic bacterium, as an etiological
agent of adult periodontal disease (White and Mayrarid, 1981; Zambn et
al., 1981; Takazoe et al., 1984; Slots and Genco, 1984; Slots et al.,
1986). For example, relatively high proportions of B. gingivalis have
been isolated from adult periodontitis lesions (Slots, 1977; Tanner et
al., 1977; Spiegel et al., 1979), patients with adult periodontitis
have been found to have higher levels of IgG antibodies to B.
gingivalis than do normal adults (Mouton et al., 1981; Naito et al.,
1984), and local immunity to B. gingivalis is greater in the more
advanced cases than in the early forms of periodontal disease (Kagan,
1980). B. gingivalis also appears to be a causative agent of
experimental periodontitis in animals (Crawford et al., 1977; Slots and
Hausmann, 1979). In addition, B. gingivalis possesses a variety of
suspected virulence factors such as proteases, collagenases,
immunologlobuiin degrading enzymes, and adhesins (Slots and Genco,
1984).
Previous investigations of Bacteroides pathogenic mechanisms have
employed the isolation and purification of B. gingivalis constituents by
12


13
B. gingivalis is the predominant bacterial species isolated from
periodontal lesions of patients with severe adult periodontitis
(Slots,1977; Tanner et al., 1977). Patients with adult periodontitis
have been found to have higher levels of IgG antibodies to B. gingivajis
than normal adults (Mouton et al., 1981) and local immunity to B.
gingivalis is greater in the more advanced cases than in the early
forms of PD (Kagan, 1980). Serum antibody titers to B. gingivalis have
been reported to decrease after therapy of adult periodontitis
patients, suggesting that antibodies to B. gingivalis result from
infection of this organism (Tolo et al., 1982). B. gingivalis is also
the most interesting and potentially virulent bacterium cultivable from
the subgingival crevice with respect to its capacity for breakdown of
tissues and host defense mechanisms (Mayrand and McBride, 1980; Van
Steenbergen et al., 1982; Nilsson et al., 1985). In addition, B.
gingivalis appears to be a causative agent of experimental
periodontitis in animals. When B. gingivalis is implanted as the
monocontaminant in gnotobiotic rats, it causes accelerated alveolar
bone loss (Crau'ford et al., 1977). In a longitudinal study of alveolar
bone loss in Macaca arctoides (Slots and Hausmann, 1979), the proportion
of B. gingivalis-type isolates reportedly increased from a minority of
the cultivable microbiota prior to bone loss to a majority of the
microflora when alveolar bone loss was detectable.
Pathogenicity of B. gingivalis
Although B. gingivalis has been strongly implicated as an
etiological agent of adult periodontitis, its exact role in the disease
process has not yet been established. In order to produce FD, it is


14
Materials and Methods
Bacteria] Strains, Plasr d and Growth Conditions
Bacteroides gingivaJis 381 obtained from a stock culture was grown
on plates containing Trypticase soy agar (BBL Microbiology Systems,
Cockeysville, Md.) supplemented with sheep blood (5%), hemin (5
micrograms per ml), and menadione (5 micrograms per ml). The organism
was also grown in 10 ml of Todd-Hewitt. broth (BBL) supplemented with
hemin (5 micrograms per ml), menadione (5 micrograms per ml) and
glucose (2 milligrams per ml). Cultures were incubated in an anaerobic
O
chamber in a N2-H2-CO2 (85:10:5) atmosphere at 37 C until the log
phase of grou'th was obtained. The 10 ml broth culture was transferred
into 25 ml of the same medium and subsequently transferred to 500 ml of
O
medium. Incubation was at 37 C anaerobically until a late log phase
culture w'as obtained. E. coli JM 109 (rec Al, end Al, gyr A96, thi,
hsd R17 sup E44, relAl, A(lac-pro AB), CF;tra D36, proAB, lac IZ M15])
and the plasmid expression vector pUC 9 (Figure 1) were gifts of J.
Messing and have been described previously (Vieira and Messing, 1982;
Yanisch-Perron et al., 1985). E. coli JM 109 uras cultured in Luria-
Bertani (LB) medium consisting of Bacto-tryptone (10 g per liter),
Bacto-yeast extract (5 g per liter), and NaCl (5 g per liter). For
solid media. Bacto-agar uras added at a final concentration of 15 g per
liter. E. coli JM 109 transformants were selected and maintained on LB
plates containing 50 micrograms of ampicillin per ml.


Figure 1
Map of pUC 9.


16
Hind III Pst I Sal I Bam HI Sma I Eco Rl
432 424 418 412 407 402
pUC 9 (2671 base pairs)


17
Preparation o Chromosomal DNA from B. giiigivaHs
Chromosomal DMA from P. gingivalis 381 was prepared by the method
of A. Das (personal communication) as follows: one to three liters of
cells were pelleted by centrifugation and washed once with lx SSC
buffer (0.87% NaC'l, 0.04% Na citrate) containing 27% sucrose and 10 mM
EDTA. The cells were pelleted and resuspended in 1/50 of the original
volume of the same buffer at 4C. Lysozyme (5 mg/ml) in SSC was added
o
to 0.5 mg/ml, the mixture was mixed thoroughly and incubated at 37 C
for 10 minutes. Nine volumes of lx SSC containing 27% sucrose, 10 mM
EDTA and 1.11% SDS (preu:arrned to 39 C) were added and the cell
O
suspension was incubated at 37 C for 10 to 30 minutes until cell lysis
was complete. In order to denature any contaminating proteins,
proteinase K was added to a final concentration of 1 mg/ml and the
lysate u'as incubated at 37 C for 4 hours. DNA was extracted twice with
phenol, twice with phenol-chloroform (1:1 by volume), and four times
with chloroform. Tu^o volumes of absolute alcohol u'ere added and the
precipitated DNA was spooled onto a glass rod. The purified DNA was
rinsed w-ith 70% ethanol and suspended in TE buffer, pH 8.0 (10 mM
Tris-HCl pH 8.0, 1 mM EDTA).
Isolation of Plasmid DNA
Plasmid DNA was isolated by the method of Ish-Horowicz and Burke
(1981) in which cells wrere lysed with SDS-EDTA in the presence of NaOH.
Potassium acetate, pH 4.8, w-as added at 4C and cell debris, protein,
RNA, and chromosomal DNA w?ere removed by centrifugation. The plasmid
wras precipitated with 2 volumes of ethanol, washed with 70% ethanol,
dried, and resuspended in TE buffer at pH 7.5. The plasmid was


18
separated from contaminating RNA and any remaining chromosomal DNA by
cesium chloride density centrifugation in the presence of ethidium
bromide. Ethidium bromide and cesium chloride were removed by butanol
extraction and dialysis, respectively. The dialyzed plasmid was then
phenol- chloroform extracted, ethanol precipitated, and resuspended in
TE buffer.
Construction of Genomic Libraries
Purified B. gingivalis DNA was partially digested with Sau 3A
restriction endonuclease to create fragments of 2-10 kilobases which
were ligated to the dephosphorylated Bam HI site of vector pUC 9 with
T4 DNA ligase by standard methods (Maniatis et al., 1982). Genomic
fragments were also obtained by partial digestion of the chromosomal
DNA with Hind III restriction endonuclease and ligated to the
dephosphorylated Hind III site of pUC 9. The recombinant plasmids were
used to transform E. coli JM 109 by the method of A. Das (personal
communication). Briefly, E. coli JM 109 was grown to an early log
phase (OD550 = 0.2) in LB broth. Ten ml of the culture were
O
centrifuged at 5,000 rpm for 5 minutes at 4 C and resuspended in 2 ml
of transformation buffer 1 (TFM 1, 10 mM Tris-HCl, pH 7.5, 0.15 M
NaCl). The cells were then pelleted and resuspended in 2 ml of TFM 2
(50 mM CaCU) and incubated on ice for 45 minutes. The cells were
again pelleted and gently resuspended in 3 ml of TFM 2, and dispensed
into 0.2 ml aliquots. One tenth ml of TFM 3 (10 mM Tris-HCl, pH 7.5,
50 mM CaCl2, 10 mM MgSO varying amounts of DNA. The cells were then allowed to incubate on ice
O
for 45 minutes, and heat shocked at 37 C for 2 minutes. LB broth (0.5


19
ml) was added and the cell suspension was incubated at 07 C for 1
hour. Finally, the cells were plated on LB agar containing ampicillin
(50 micrograms per ml) and 5-bromo-4-chloro-3-indolyl- 8-D-
galactopyranoside (X-Gal) (200 micrograms per ml) and incubated for 24
O O
to 48 hours at 37 C. All transformants were stored at -70 C in LB
broth wth ampicillin (50 micrograms per ml) and 20% glycerol.
Preparation of Antisera
Late exponential phase cells of B. gingivalis strain 381 were
pelleted, washed wth 0.01 M phosphate-buffered saline (PBS) pH 7.2,
O
and resuspended in PBS and 0.01% sodium azide at 4 C for at least 1
hour. The cells were again washed with PBS, resuspended to a
concentration of 1 x 109 cells per ml and emulsified in an equal
volume of Freund's incomplete adjuvant. The cell emulsion was injected
in 3 doses at two wreek intervals for 4 weeks subcutaneously in the back
of adult New Zealand rabbits. Each rabbit was given a booster dose 50
to 60 days later. Antisera were collected from the marginal ear veins
just prior to immunization and beginning one week after the booster
O
dose. All sera were stored at -20 C.
Rabbit anti-B. gingivalis antiserum was adsorbed 4 times wth E.
coli JM 109 harboring pUC 9 plasmid E. coli JM 109 (pUC 9) For
each adsorption, E. coli cells from 1 liter of a stationary phase culture
O
were washed and mixed with 3 ml of serum at 4 C for 1 hour. The serum
was recovered by pelleting the cells at 5,000 x g for 20 minutes. For
sonicate adsorption, E. coli cells from 500 ml of stationary phase growth
suspended in 5 ml of PBS were disrupted by sonication and mixed with E.
O
coli cell-adsorbed serum for 1 hour at 4 C. The mixture was centrifuged


20
at 100.000 x g for 1 hour and the resulting dear serum was stored at
O
-20 C.
Assay of Antibody Titer
Sera were tested for anti-13, gingivalis and anti-E. coll activities
by an enzyme-linked immunosorbent assay (ELISA). B. gingivalis
cells suspended in carbonate-bicarbonate buffer. pH 9.6, (108 cells
O
per well) were fixed to microtiter plates at 4 C overnight. After the
wells were washed with 0.5% Tween 20 in PBS, 1% bovine serum albumin
(BSA) in PBS was added to each well, and the plates were incubated for
2 hours at room temperature in order to saturate the binding sites.
After washing the plates, serially diluted antiserum w:as added and
plates were incubated for 1 hour at room temperature followed by a
second wash with 0.5% Tween 20 in PBS. Peroxidase conjugated goat
anti-rabbit IgG, diluted 1:1000 in 1% BSA, w?as added and the plates
were again incubated at room temperature for 1 hour. After a final
washing, a color-forming substrate solution (O-phenylenediamine, 0.5 g
per 100 ml in 0.1 M citrate buffer pH 4.5 and 1.8% hydrogen peroxide)
was added, and the plates were incubated for 30 minutes at room
temperature. The absorbance at 492 nm wras measured with a Titertek
Multiscan reader. An absorbance of 0.05 or more over background was
considered positive. Background readings were obtained from the wells
in which all reagents except anti-5. gingivalis antiserum was added.
Normal rabbit serum was also tested against B. gingivalis antigen.
To test the effectiveness of adsorption, the titers of treated
sera w'ere assayed as described above except that E. coli JM 109 (pUC9)
whole cells were used as the antigen.


21
FilterBinding Enzyme immunoassay
Ampicillin-resistant transformants which formed white colonies in
the presence of X-Gal were spotted onto LB agar plates with arnpicillin,
grown overnight, and blotted onto nitrocellulose filter disks. B.
gingivalis and E. coli JM 109 (pUC 9) were also spotted onto each
filter as a positive and negative control, respectively. Duplicate
prints of the colonies on nitrocellulose filters were made and colonies
on one of each duplicate print were lysed by a 15-min. exposure to
chloroform vapor. Filters were then air dried for 30 minutes and
soaked for 2 hours in PBS containing 3% bovine serum albumin. After the
filters were urashed, adsorbed rabbit anti-5. gingivalis antiserum tpas
added and the filters were incubated in a solution of peroxidase
conjugated goat anti-rabbit immunoglobulin for 1 hour. After washing,
the filters were developed in a color-forming substrate solution
consisting of 0.06% 4-chloro-l- naphthol and 3% hydrogen peroxide in a
1:4 solution of methanol-TBS (50 mM Tris hydrochloride, 200 mM NaCl, pH
7.4). Clones which developed a blue color w-ere picked and rescreened
by the same procedure.
Restriction Analysis of Recombinant Plasmids
Plasmids were isolated from all the clones that were positive in
the filter-binding enzyme immunoassay. Restriction endonuclease
digestions were performed under conditions described by the
manufacturer to produce complete digestion. Agarose gel
electrophoresis was performed as described by Maniatis et al. (1982).
The size of DNA bands was estimated by comparing the distance of
migration to a logrithmic plot of the migration of standard restricted


22
lambda DNA run on the same gel.
Southern Plot Analysis
Recombinant plasmid and pUC 9 vector DNAs were digested to
completion with the appropriate restriction enzymes and run on a 1.2%
agarose gel. B. gingivalis DNA partially digested with Sau 3A, and Hind
III digested Eikenella corrodens clone 18 DNA (unpublished) were
also loaded in the gel. The DNA was transferred to Biodyne nylon
membrane by Southern transfer (Southern, 1975). B. gingivalis DNA
partially digested with Hind III was nick translated with ( a -32P
dCTP) (400 Ci/mmol, Amersham Corp., Arlington Heights, Ill.) as
described by Maniatis et al. (1982). The membrane-bound DNA w'as
O
hybridized to the nick-translated probe at 42 C in 50% formamide for 16
hours by the method recommended by the manufacturer (Pall Ultrafine
Filtration Corp., Glen Cove, N.Y.) which adapted from Wahl et al.
(1979). The membrane wras washed at room temperature in wash buffer (2
O
x SSC and 0.1% SDS) four times each for 5 minutes and twice at 50 C
each for 15 minutes in O.lx SSC, 0.1% SDS. An autoradiogram vras
obtained wth Kodak XAR-5 film (Eastman Kodak Co., Rochester, N.Y.)
and Cronex Quanta II intensifying screen (Du Pont Co., Wilmington,
Del.).
Assay of the Titer of Ariti-ff. gingivalis Antiserum to E. coli
Transformants Which Express B. gingivalis Antigens
Cultures of each representative clone were prepared by 100 fold
O
dilution of overnight cultures and grown for 2 hours at 37 C.
Isopropyl- 6 -D-thiogalactopyranoside (IPTG) was added to specific


23
cultures at a final concentration of 1 rnM and the ce-ls were pelleted
by centrifugation 4 hours later. The cells were washed, resuspended in
1/10 volume of PBS, and the optical density of each suspension was
determined at 550 rim. Cell lysate antigen was prepared by breaking the
cells with a sonicator. The protein concentration of each lysate was
determined by the Bio-Rad protein assay (Bio-Rad Laboratories,
Richmond, Calif.). Determination of the titer of anti-5, gingivalis
381 against these antigens was performed with the ELISA as described
above (10B cells or 1 pg protein per well). Normal rabbit serum
exhaustively adsorbed with E. coli JM109 (pUC9) was also tested in the
same manner.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and Western Blot Analysis of the Expressed Antigens
Each of the representative antigen-producing clones was grown to
mid-log phase in 3.0 ml of LB broth with 50 micrograms of ampicillin
per ml. The cells were pelleted, washed with PBS, resuspended in 0.3
ml of sample buffer (62.5 mM Tris-hydrochloride, 5% 2-mercaptoethanol,
2% SDS, 10% glycerol, 0.002% bromophenol blue, pH 6.8), and boiled for
3 minutes. The B. gingivalis cell lysate was mixed with an equal
volume of sample buffer and treated in the same manner. SDS-PAGE was
performed in a vertical slab gel electrophoresis tank (Hoefer
Scientific Instruments, San Francisco, CA.) as described by Laemmli
(1970). Samples of 0.03 ml from each clone as well as 40 micrograms of
Bacteroides cell lysate were run at a constant current of 20 mA per gel
through the 4% polyacrylamide stacking gel (pH 6.8) and 30 mA per gel
through the 12.5% or 5% separating gel (pH 8.3). The gels were


24
processed either by staining with Coomassie brilliant blue R2-"1
(Fairbanks et al., 1971), or used for Western blot analysis.
Western blotting was done as described by Burnette (1981) as follows.
Separated antigens on the gel were transferred to nitrocellulose paper
(0.45 m) (Schleicher & Schuell Co., Inc., Keene, NH) by
electroblottirig using the Hoefer apparatus at CO V overnight with a
buffer containing 20 mill Tris base, 150 mM glycine, and 20% methanol (pH
8.3). The blot was visualized as follows. Nitrocellulose sheets were
preincubated in a blocking solution of PBS with 2% BSA and 0.1%
Tween-20 for 2 hours or overnight. Adsorbed antisera used as probes
were usually diluted 1:100 in blocking solution and reacted with the
nitrocellulose transfer for 1.5 hours. After w'ashing with distilled
water, the membranes were incubated with affinity purified goat
anti-rabbit IgG for 1.5 hours. After washing again with distilled
water, the membranes w^ere developed in the color-forming substrate
solution as described in the filter-binding enzyme immunoassay. The
molecular weight of each individual band wras estimated by comparison to
the molecular weight standard proteins run on the same gel.
Results
Titer of Antisera
Rabbit anti-B. gingivalis antiserum had an antibody titer of 1:
64,000 to B. gingivalis and 1:160 to E. coli (pUC 9), whereas
normal rabbit serum had an antibody titer of 1:10 to B. gingivalis and
1:80 to E. coli (pUC 9). Adsorption of anti-B. gingivalis antiserum
with E. coli (pUC 9) resulted in a slight reduction of antibody titer


25
to B. gingivalis and reduced the anti-s', coli titer to zero or 1:10.
Identification of E. coli Tra:¡sformants Which Expressed B. gingivalis
Antigens
Approximately 4,500 tranformants generated from the Sau 3A restricted
chromosomal DNA were tested for the expression of B. gingivalis
antigens by the filter-binding enzyme immunoassay using E. coli-
adsorbed rabbit anti-B. gingivalis serum. Only 1 clone (clone 3)
was positive when either lysed or unlysed cells were tested. A total
of 1,700 colonies of transformants resulting from Hind III restricted
chromosomal DNA were also tested for the expression of B. gingivalis
antigens. Seven clones gave positive signals. Of these 7 clones, one
uTas positive only when lysed (clone 8) and the rest were positive both
when lysed and unlysed (Table 1).
Agarose Gel Electrophoresis of Recombinant Plasmids
To further confirm the positive results of the filter-binding
enzyme immunoassay, plasmid DNA was isolated from each positive clone.
Electrophoresis of these unrestricted plasmids showed that each clone
contained only one recombinant plasmid (Figure 2, lanes 1 through 8).
Clone 3, which was constructed by ligation of Sau 3A partially
digested B. gingivalis DNA with Bam HI cut pUC 9, could not be digested
with Bam HI (Figure 3, lane 10). Restriction of pUC 9 with enzyme Sma
I and Sal I deletes a 9 bp fragment containing the Bam HI site from pUC
9 (Figure 2, lane 18 and Figure 3, lane 4, see Figure 1 for map of pUC
9). Therefore, clone 3 DNA w-as restricted with Sma I and Sal I.
Restriction analysis revealed a fragment of linear 9 bp-deleted pUC 9


26
Table 1. Characterization of E. coli transformants
which express B. gingivalis antigens
Colonies reacted
Size of B.
Clone No.
with antiserum
unlysed lysed
gingivalis
DNA cloned (Kb)
1 and 2
+ a
+
3.2
3
+
+
1.1
4
+
+
3.3
5 and 6
+
+
5.5
7
+
+
4.8
8
b
+
3.5
a = Positive reaction
b = Negative, not reactive


Figure 2. Agarose gel electrophoresis of recombinant plasmids.
Lanes: 1-8, undigested recombinant plasmids from clones 1 8; 9, pUC 9
digested with Hind III; 10 13, recombinant plasmids from clones 5, 6, 7, and 8
digested with Hind III; 14, pUC 9 digested with Pvu II; 15 17, recombinant
plasmids of clones 1, 2, and 4 digested with Pvu II; 18, pUC 9 digested with Sma
I and Sal I; 19, recombinant plasmid of clone 3 digested with Sma I and Sal I.


28


29
and 2 fragments of insert (Figure 2, lane 19 and Figure 3, lane 5).
Restriction analysis with different enzymes (Figure 3) showed that the
size of insert of clone 3 was approximately 1.1 kb.
Clones 1, 2, 4, 5, 7, and 8 were generated from Hind Ill-
restricted chromosomal DNA. After digestion with Hind III, only clones
5, 6, 7, and 8 revealed fragments of the linear pUC 9 vector and
fragments of B. gingivaJis DNA inserts (Figure 2, lanes 10 through 13).
Plasmid DNAs of these clones were restricted with various enzymes and
analyzed by gel electrophoresis (Figure 4). The estimated siz- of
inserts of clones 5, 6, 7, and 8 are 5.5, 5.5, 4.8, and 3.5 kb,
respectively (Table 1). Thus clones 5 and 6 were found to contain
plasmids of the same size and identical restriction fragments.
Although clones 1, 2, and 4 were generated from Hind III
restricted DNA, they did not result in fragments of linear pUC 9 after
Hind III digestion (Figure 5, lanes 6, 11, and 16). These cloned DNAs
were then restricted with Pvu II, which generates a 307 bp fragment
containing the polylinker-cloning sites from pUC 9 (Figure 1 and Figure
2, lane 14 and Figure 5, lane 4). Clones 1, 2, and 4 revealed
fragments of linear 307 bp-deleted pUC 9 and inserts associated with
the deleted fragment (Figure 2, lanes 15, 16, and 17). These cloned
DNAs were digested wdth various restriction enzymes and analyzed by
agarose gel electrophoresis (Figure 4). The size of inserts of clones
1, 2, and 4 were estimated to be 3.2, 3.2, and 3.3 kb, respectively
(Table 1). Clones 1 and 2 also contained plasmids of the same size and
identical restriction fragments.


Figure 3. Agarose gel electrophoresis of different restriction digests of the
recombinant plasmid from clone 3.
Lanes: 1, DNA marker-Hind 111/Eco RI digest of lambda DNA; 2, undigested pUC 9;
3, pUC 9 digested with Hind III; 4, pUC 9 digested with Sma I and Sal I; 5, 6, 7,
8, 9, and 10, recombinant plasmid from clone 3 digested with Sma I and Sal I, Sma I
alone, Sal I alone, Hind III, Eco RI, and Bam HI, respectively; 11, undigested
recombinant plasmid from clone 3; 12, DNA marker-Hind III digest of lambda DNA.




Figure 4. Agarose gel electrophoresis of different restriction digests of
recombinant plasmids from clones 5, 6, 7, and 8.
Lanes: 1, DNA marker-Hind III/Eco RI digest of lambda DNA; 2, pUC 9 digested
with Hind III; 3, 4, and 5 recombinant plasmid from clone 5 digested with Hind III,
Eco RI and Bam HI respectively, 6, 7, and 8, recombinant plasmid from clone 6 digested
with Hind III, Eco RI and Bam HI, respectively; 9, 10, and 11, recombinant plasmid
from clone 7 digested with Hind III, Eco RI and Bam HI, respectively; 12, 13, and 14,
recombinant plasmid from clone 8 digested with Hind III, Eco RI and Bam HI,
respectively; 15 18, undigested recombinant plasmids from clones 5-8; 19, DNA
marker-llind III digest of lambda DNA.




Figure 5. Agarose gel electrophoresis of different restriction digests of
recombinant plasmids from clones 1, 2, and 4.
Lanes: 1, DNA marker-Hind III/Eco RI digest of lambda DNA; 2, pUC 9 undigested;
3, pUC 9 digested with Hind III; 4, pUC 9 digested with Pvu II; 5, 6, 7, and 8,
recombinant plasmid from clone 1 digested with Pvu II, Hind III, Eco RI and Bam HI,
respectively; 9, undigested recombinant plasmid from clone 1; 10, 11, 12, and 13,
recombinant plasmid from clone 2 digested with Pvu II, Hind III, Eco RI, and Bam HI,
respectively; 14, undigested recombinant plasmid from clone 2; 15, 16, 17, and 18,
recombinant plasmid from clone 4 digested with Pvu II, Hind III, Eco RI, and Bam HI,
respectively; 19, undigested recombinant plasmid of clone 4.




36
Hybrid nation of Recombinant Plasmids rith D. gingivalis DXA Probe
Southern t iot analysis was also performed to confirm that the DNA
inserts were derived from the P. gingivalis DNA. As can be seen in
Figure 6, the hybridization pattern of most of the insert fragments
showed dark bands of homology to the B. gingivalis chromosomal DNA
probe. The pUC 9 showed a faint band with homology to the probe.
Increasing the stringency of the wash (65 C for 1 hour) did not
significantly change the hybridization pattern. How'ever, a shorter
exposure of the autoradiograph eliminated the background of pUC 9 but
the two smallest insert bands from clone 4 also disappeared. The
control DNA from Eikenella corrodens did not hybridize with the P.
gingivalis DNA probe (Figure 6, lane 12).
Titer of Anti-fl. gingivalis Antiserum to E. coli Transformants
Anti-B. gingivalis antiserum was able to detect antigen expression
in all positive clones except clone 8 in an enzyme-linked immunosorbent
assay (ELISA) (Table 2). The antiserum reacted with both whole cell
and cell lysate antigens. Isopropyl- 8-D-thiogalactopyranoside (IPTG)
was not necesary to induce antigen expression. However, in the
presence of IPTG, clones 2 and 3 showed higher antigen expression,
especially when the cell lysate preparations were tested.
Determination of the Expressed Antigens in E. coli JM 109
Five stable representative clones were analyzed for antigen
expression by SDS-PAGE and Western blot analysis. As can be seen in
Figure 7, only clones 2 and 3 produced antigens detectable by E. coli
adsorbed anti-fh gingivalis antiserum in the Western blot. Antigens


32
Figure 6. Hybridization of recombinant plasmids with P labeled B. gingivalis
DNA probe.
A. Agarose gel electrophoresis of DNA before Southern transfer.
Lanes: 1, Sau 3A partially digested B. gingivalis DNA; 2, pUC 9 digested with Pvu II;
3, recombinant plasmid from clone 1 digested with Pvu II; 4, recombinant plasmid from
clone 2 digested with Pvu II; 5, recombinant plasmid from clone 4 digested with Pvu II;
6, pUC 9 digested with Hind III; 7, recombinant plasmid from clone 5 digested with Hind
III; 8, recombinant plasmid from clone 6 digested with Hind III; 9, recombinant plasmid
from clone 7 digested with Hind III; 10, recombinant plasmid from clone 3 digested with
Sma I and Sal I; 11 and 12, recombinant plasmid from Eikenella corvodens clone 18
digested with Hind III.
B. Autoradiograph of Southern blot hybridization of the agarose gel in panel A.


38


39
Table 2. Titer of anti-5, gingivalis antiserum against E. coli
transformants which express B.gingivalis antigens
Antibody titers
against test antigensb
Organism
Whole cell
Cell lysate
IPTG- IPTG+
IPTG- IPTG+
Clone 1
320
NTC
320 640
NT
2
320
640
320 640
1,280 2,560
3
20
160
40 160
1,280
4
20 100
20 40
20 40
20 40
5
40 80
40 80
40 80
40 80
6
40
NT
40
NT
7
40
40
40
40
8
0
0
0
NT
E. coli
JM 109 (pUC
9) 0-10
0-10
0-10
0-10
B. gingivalis
40,950 64,000
NT
NT
NT
Control NRSd
a Number designates the reciprocal dilution of the sera which gave
OD492 reading of 0.05 or more over the background. Antiserum was
exhaustively adsorbed with E. coli JM109 (pUC 9).
b Antigens were prepared from cultures grown without IPTG (IPTG~)
or in the presence of IPTG (IPTG+>-
c Not tested.
d Normal rabbit serum exhaustively adsorbed with E. coli JM 109
(pUC 9) did not react to test antigens.


Figure 7. SDS-PAGE on 12.5% acrylamide and Western blot analysis of expressed
B. gingivalis antigens. Molecular weight standards (Pharmacia Fine Chemicals,
Piscataway, N.Y.) are phosphorylase b (94 K, 94,000 molecular weight), albumin
(67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor
(20,100) and a-lactalbumin (14,400).
Lanes: 1, B. gingivalis cell lysate (40 ng); 2 to 7, whole cell samples of clones
2, 3, 4, 5, 7, and E. coli JM 109 (pUC 9) as described in the methods.
A. The gel after Coomassie blue stain. B. The blot was probed with E. ooli-
adsorbed anti-B. gingivalis antiserum. C. The blot was probed with normal
rabbit serum.




o
expressed in clones 4, 5, and 7 were not detected by Western blot
analysis. Normal rabbit serum reacted to some common antigens among
these clones and E. coli JM 109 (pUC 9). The anti-B. gingjvalis
antiserum did, however, react with a protein band of approximately
140,000 (140 K), as well as a smear of lower molecular weight from
clone 2. Multiple bands of 30 to 50 K from clone 3 were also detected.
These particular polypeptides were not detectable in E. coli JM 109
(pUC 9) preparations (Figure 7, lane 7). A whole cell preparation from
clone 2 was also separated in a 5% SDS polyacrylamide gel and the
expressed protein w'as estimated to have a molecular u'eight of 125 K
(Figure 8).
Discussion
Genomic libraries of B. gingivalis DNA were constructed in the
plasmid expression vector pUC 9, which contains the pBR 322 origin of
replication, the pBR 322 ampicillin resistance gene, and a portion of
the lac Z gene of E. coli which codes for the a -peptide of
8-galactosidase (Figure 1). The amino terminus of the lac Z gene
contains a polylinker region wrhich has multiple unique cloning sites.
Transformation of E. coli JM 109, which is defective in 8-
galactosidase, with pUC 9 complements the bacterial 8 -galactosidase
activity, resulting in the ability of the bacterial cell to metabolize
the lactose analog X-Gal to a blue color. Cloned DNA inserted in the
polylinker region will interrupt the lac Z gene of the plasmid.
Therefore E. coli transformants resulting from recombinant plasmids will
be unable to metabolize X-Gal and appear as u'hite colonies on X-Gal
containing plates. The advantages to this plasmid are 1) DNA inserted


Figure 8. SDS-PAGE (on 5% acrylamide) of expressed B. gingivalis
antigen in clone 2.
Lanes: A) Molecular weight standards (Sigma Chemical Co., St.
Louis, Mo.) are myosin (205 K, 205,000), g-galactosidase (116,000),
phosphorylase B (97,400); B) Whole cell sample of clone 2.


44
205 K


45
into any of the cloning sites, which are downstream from a strong
promoter, should be expressed whether or not a B. gingivalls promoter
is cloned with a structural gene, 2) transformants containing a
recombinant plasmid are easily detected upon initial selection, and 3)
the multiple cloning sites make it a versatile cloning vector which is
especially useful for subcloning.
Five different E. coii clones stably exhibited B. gingivaiis
antigen expression. These antigens were detected in intact cells both
by filter-binding enzyme immunoassay (Table 1) and ELISA (Table 2).
Although it has not yet been confirmed by immunoeleetronmicroscopy, it
is likely that these Bacteroides antigens are located on the E. coli
cell surface, and therefore must contain a leader peptide in order to
be translocated to the E. coli surface (Oliver, 1985). This result
suggests that B. gingivaiis surface antigens can be processed as well
as expressed in E. coli.
Clones 1 and 2 have undergone some kind of DNA rearrangement,
i.e., the recombinant plasmids, 'when cut by Hind III, did not result
pUC 9 and insert band but showed one large band and one small band
(Figure 2, lanes 10 and 11). This apparent rearrangement may result
from a deletion at one Hind III end of the insert and another Hind III
end may still be intact.
Clone 2 w-as found to encode a polypeptide with an average
molecular weight of 125 K, seen in polyacrylamide gels and detected by
Western blot analysis (Figures 7 and 8). The smear at the lower
molecular weight seen in the blot may be the degraded product of this
expressed antigen, since E. coli has a functional Ion gene which
encodes for the enzyme involved in degradation of internal abnormal


46
proteins (Charette et al., 1981; Chung and Goldberg, 1981; Waxman and
Goldberg, 1982).
The function of the lac promoter in pUC 9 does not depend on IPTG;
it is, however, enhanced by IPTG, since E. coli JM 109 (pUC 9) grows as
blue colonies on medium containing X-Gal in the absence of IPTG.
Expression of the B. gingivalis antigen in clone 2 occurs either in the
presence or absence of IPTG but is enhanced by IPTG stimulation. This
result suggests that the direction of transcription of this DNA insert
is the same as that of g-galactosidase and is likely to be under the
control of the lac promoter. Assuming an average molecular weight of
100-125 for an amino acid, the insert of clone 2, estimated to be
3,200 bp, could encode for a 125 K polypeptide. The expressed
polypeptide may be fused to the major portion of the ot-peptide of
8-galactosidase which would add approximately 100 amino acids to the
expressed polypeptide. The expression of the clone 3 antigen was also
found to be stimulated by IPTG in the same manner as clone 2. The
size of the clone 3 insert (1.100 bp) is large enough to encode for the
expressed antigen (30 to 50 K) observed by Western blotting.
The synthesis of Bacteroides antigens in clones 4, 5, and 7 was
not found to depend on the presence of IPTG or to be enhanced by IPTG
(Table 2). This suggests that a functional Bacteroides promoter is
included with the structural gene of each clone. However, antigen
expression of these clones cannot be detected by Western blot
analysis. This might be due to 1) the antigens not being transferred
to the nitrocellulose sheets, 2) the transferred antigens containing
altered conformations which are not recognized by the antiserum, or 3)
the antigen expression being too low to be detected.


47
These results have demonstrated that the B. gingivalis genome
can be cloned and expressed in E. coli. The cloned antigens are
presently being identified and further characterized for functional
properties. The cloning of B. gingivalis genes is an approach that
provides new tools for investigations into the pathogeneeity of B.
gingivalis.


CHAPTER THREE
CHARACTERIZATION OF BA CTEROIDES GINGIVALIS
ANTIGENS SYNTHESIZED IN ESCHERICHIA COLI
Introduction
Bacteroides glngivalis possesses several potential virulence
factors which may 1) promote its colonization in the host, 2) resist
host defenses, and 3) cause destruction of periodontal tissues (Slots
and Genco, 1984). Colonization, the initial event in the establishment
of disease, requires the adherence of bacteria to host tissues (Gibbons
and Van Houte, 1975), therefore bacterial surface components which
mediate bacterial adherence are considered to be important virulence
factors. In the oral cavity, bacteria can attach to host tissues as
well as to bacteria in pre-formed plaque (Slots and Gibbons, 1978).
The nature of the binding sites on teeth and oral tissues to u'hich
periodontopathic bacteria, including B. gingivalis, attach has not been
well established. In vitro, B. gingivalis can attach to and
agglutinate erythrocytes (Okuda and Takazoe, 1974; Slots and Gibbons,
1978; Slots and Genco, 1979; Okuda et al., 1981), can adhere in high
numbers to human buccal epithelial cells (Slots and Gibbons, 1978;
Okuda et al., 1981), to crevicular epithelial cells derived from
periodontal pockets (Slots and Gibbons, 1978), and to surfaces of Gram
positive bacteria present in plaque, (Slots and Gibbons, 1978; Schwarz
et al., 1987). In addition it will adhere to untreated and saliva-
treated hydroxyapatite (SHA), but in comparatively low numbers (Slots
and Gibbons, 1978). B. gingivalis has also been reported to bind to
48


49
HR9 matrix, a material similar to the basement membrane barrier
underlying connective tissue (Leong et al., 1985). Recently, it has
been reported that B. gingivalis can bind to fibrinogen and possibly
colonize host tissue by attaching to fibrinogen-coated surfaces (Lantz
et al., 1986).
Since the components involved in B. gingivalis adherence in vivo
are, at present, ill defined, the expression of any structure detected
by in vitro methods thus needs to be examined. Therefore, the
antigen-expressing clones described in Chapter Two were tested for the
expression of adhesins for saliva-treated hydroxyapatite (SHA adhesin)
and erythrocytes (hemagglutinin). This chapter describes the assay for
the SHA adhesin by testing for removal of SHA adherence inhibition by
anti-B. gingivalis antiserum and the assay for hemagglutinin by a
direct hemagglutination test. The clones which were able to
agglutinate erythrocytes 'were analyzed by restriction analysis of their
B. gingivalis DNA inserts and DNA homologies were tested by Southern
blot hybridization. Antibodies against these clones were made in
rabbits and used as probes to identify the native antigens of B.
gingivalis by Western blot hybridization. B. gingivalis DNA inserts
from clones 2 and 7 were used as probes in the hydridization of several
restricted B. gingivalis chromosomal DNAs to determine whether these
inserts are adjacent to each other in the chromosomal DNA.


50
Materials and Methods
Bacterial Strains and Growth Conditions
Bacteroides gingivalis 381 was cultured in Todd-Hewitt broth as
described in Chapter Two. E. coli transformants were cultured in LB
medium containing 50 micrograms of ampicillin per ml by preparing 100
fold dilutions of overnight cultures followed by incubation for 2 hours
O
at 37 C. IPTG was added to the cultures, when used at a final
concentration of 1 mM, and the cultures were incubated for an
additional 4 hours.
Assay for Removal of SHA Adherence Inhibition by Anti-13. gingivaJis
Antiserum
Aliquots of anti-B. gingivalis antiserum were adsorbed with each
antigen-expressing clone as well as E. coli JM 109 (pUC 9) as described
in Chapter Two. The titer of each adsorbed antiserum was tested
against ee h clone and B. gingivalis whole cell antigen by ELISA as
described above.
Whole paraffin-stimulated human saliva was collected and heated at
O
56 C for 30 minutes to inactivate degradative enzymes. Extraneous
debris and cells were removed by centrifugation at 12,000 rpm for 10
minutes and sodium azide w'as added to a final concentration of 0.04%.
Hydroxyapatite beads (HA) (BDH Biochemical, Ltd., Poole, England)
urere treated as previously described (Clark et al., 1978). Briefly, 10
mg of beads were washed and hydrated in distilled water in 250
microliter plastic microfuge tubes followed by equilibration overnight
with adsorption buffer (0.05 M KC1, 1 mM K2HPO4, pH 7.3, 1 mM CaCl2


51
and 0.1 mM MgCh). The beads were incubated with 200 microliters of
saliva for 24 hours at 4C and then washed with sterile adsorption
buffer to remove nonadsorbing material. Control tubes without HA were
treated identically.
B. gingivalis 381 cells were labeled by growth to late log phase
in medium supplemented with (3H) thymidine (10 mCi/ml). The cells
were pelleted, washed twice in adsorption buffer, and dispersed with
three 10-second pulses (medium power) with a microultrasonic cell
disrupter.
The dispersed cells were mixed with each antiserum (1:100
dilution) and normal rabbit serum to a final concentration of 4 x 106
cell/ml. The cell-antiserum suspensions (200 microliters) wrere then
added to the SHA beads in microfuge tubes and the tubes were rotated in
an anaerobic chamber for 1 hour. Labeled cells alone (no antisera)
were treated in the same manner to determine the number of cells
adhering to the SHA surface. A control tube containing cells but no
SHA wras tested to quantitate the amount of cells bound to the tubes
rather than to the SHA. One hundred microliters of adsorption buffer
containing unadhered cells wras removed and placed in minivials
containing 3 ml of aqueous scintillation cocktail (Amersham/Searle,
Arlington Heights, IL), and counted with a Scintillation Counter (Model
455 Parkard Tricarb). Determination of the number of cells adhering to
the SHA was done by subtracting the number of cells (no. of counts) in
solution from the total number of cells (no. of counts) which did not
adhere to the tube.


52
Direct Hemagglutination Assay
The hemagglutination assays were carried out in V-bottom
microtiter plates (Dynatech Laboratories, Inc., Alexandria, Virginia).
Erythrocytes (sheep or human group 0) were washed 3 times with PBS
(0.02 M phosphate buffered saline), pH 7.2, and resuspended to a final
concentration of 0.2% (v/v). Cells of B. gingivalis and antigen
expressing clones were washed twice in PBS and resuspended to an
optical density of 0.5 and 2.0, respectively, at 660 rim. The cell
suspensions were diluted in a twofold series with PBS and 0.05 ml of
the suspensions were added to the wells. E. coli JM 109 (pUC 9) which
was prepared in the same manner as the antigen-expressing clones, was
included as a control. An equal volume (0.05 ml) of washed
erythrocytes was added and mixed with the bacterial cells. The plates
were stored for 16 hours at 4 C and then examined for evidence of
hemagglutination as follows. Agglutinated erythrocytes will settle as
clumps which will be dispersed throughout the bottom of the wells,
resulting in a pinkish-red coating of each well. In the absence of
hemagglutination, the erythrocytes will settle on the bottom of the
well as a central, smooth, bright red round disk. The titer was
expressed as the reciprocal of the highest dilution showing positive
agglutination.
Hemagglutination Inhibition Assay
The hemagglutination inhibition assay was also carried out in V-
bottom microtiter plates. B. gingivalis cell suspensions in PBS u'ere
adjusted to the optical density of 0.5 at 660 nm. Each antiserum
examined for hemagglutination inhibition activity was diluted two-fold


53
in a series of wells. Fifty gl of the bacterial suspension with twice
the minimum number of cells which produced hemagglutination was then
added to each well. After incubation with gentle shaking at room
temperature for 1 hour, 0.05 ml of the w'ashed erythrocytes were added
to each well and mixed. The plates are left for 16 hours at 4 C and
read for hemagglutination as described above for the hemagglutination
assay. The titer was expressed as the reciprocal of the highest
dilution showing hemagglutination inhibition.
Preparation of Antisera to Hemagglutinable E. coli
E. coli transformants which were able to agglutinate erythrocytes
were grown in LB broth containing ampicillin as described above. Tw'o
rabbits w'ere injected wdth each clone as described in Chapter Two.
Sera were exhaustively adsorbed with E. coli JM 109 (pUC 9) and tested
for anti-B. ginglvalis activity by ELISA.
Adsorption of Anti-Clone 2 Antiserum
Anti-clone 2 antiserum diluted 1:10 was separately adsorbed with B.
gingivalis, E. coli JM 109 (pUC 9), and clones 2, 5, and 7. Washed
stationary phase cells of each bacterial culture were prepared as
described in Chapter Twro. For each adsorption, 107, 108, 109, and 1010
bacterial cells were mixed with 200 ul of serum and the suspensions
were stored at 4C overnight. The sera were recovered by
centrifugation at 12,000 g for 10 minutes. Each adsorbed antiserum was
assayed by ELISA to determine the titer to B. gingivalis.


54
DNA Procedures
Restriction endonuclease digestions of the recombinant plasmids
from clones 2, 5, and 7 were performed according to manufacturer's
directions. The size of DNA inserts were estimated and Southern blot
analysis was performed as described in Chapter Two. Clone 5 DNA was
digested with Hind III and two fragments of B. gingivalis inserts were
isolated from agarose gels by the method of Zhu et al. (1985) employing
centrifugal filtration of DNA fragments through a Millipore membrane
inside a conical tip. The DNA preparations were extracted with phenol-
chloroform, precipitated with ethanol and resuspended in TE, pH 8.0.
Each DNA fragment was ligated to Hind III digested pUC 9 and the
resulting recombinant plasmids were transformed into competent E. coli
JM 109 cells as described in Chapter Two. Recombinant plasmids from
these transformants were isolated by rapid plasmid DNA isolation
(Silhavy et al., 1984), digested with appropriate restriction
endonucleases, and analyzed by agarose gel electrophoresis.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and Western Blot Analysis
B. gingivalis cell lysate and cells of E. coli transformant wrere
prepared and analyzed by SDS-PAGE and Western blot techniques as
described in Chapter Two. Antisera to clones 2, 5, and 7 exhaustively
adsorbed with E. coli JM 109 (pUC 9) were used as probes in the Western
blot. Control antisera included anti-clone 2 antiserum also adsorbed
with B. gingivalis at the ratio of 1010 cells per 100 ul of antiserum,
and antiserum to E. coli JM 109 harboring pUC 9 with Eihenella
corrodens DNA insert.


55
Results
Assay for SHA Adhesin
It is possible that if the B. gingivalis SHA adhesin is expressed
in E. coli, it may be expressed in a functionally inactive form, due to
spacial interference by other E. coli surface structures, or it may not
be processed adequately by the E. coli protein translocation machinery
and thus may not be properly expressed on the E. coli surface. However,
there is a strong possibility that the expressed antigen would still be
antigenically intact. Thus, anti-15, gingivalis 381 antiserum which
inhibits the adherence of B. gingivalis 381 to SHA was adsorbed with
each antigen-expressing clone until the titer of this antiserum to each
clone was reduced to zero. Each adsorbed antiserum was tested for
inhibition of B. gingivalis adherence to SHA. If a clone expresses an
antigenically active adhesin, the adsorbed antiserum should be unable
to inhibit B. gingivalis 381 adherence to SHA or may partially inhibit
the adherence.
The results in Table 3 summarize the SHA inhibition data and
indicate that the antiserum adsorbed with each antigen-expressing clone
still inhibited the adherence of B. gingivalis. There is no apparent
significant decrease in the percent inhibition by each adsorbed
antiserum.
Assay for Hemagglutinin
The rationale to identify the clones which express hemagglutinin
were analogous to those described for the SHA adhesin. The anti-5.
gingivalis antiserum adsorbed with each antigen-expressing clone and E.


56
Table 3. Inhibition of adherence to SHA by adsorbed
anti-5, gingivalis antisera
Inhibitor and dilution
% adherence8
% inhibition15
None
83.35
Normal rabbit serum
1:100
80.08
0.05
Antiserum unadsorbed
Antiserum adsorbed with:
1:100
22.70
72.15
E. coli JM 109 (pUC 9)
1:100
21.57
73.07
Clone 2
1:100
10.73
86.59
Clone 3
1:100
22.60
71.78
Clone 4
1:100
16.24
79.71
Clone 5
1:100
27.37
65.82
Clone 7
1:100
19.90
75.15
a Percent adherence was calculated from the following formula:
% adherence = [ (CPM from tube without SHA CPM from tube with
SHA)/(CPM from tube without SHA)] x 100.
b Percent inhibition was calculated from the following formula:
% inhibition = Cl (% adherence in the presence of antibody / %
adherence in the absence of antibody)] x 100.


57
coli JM 109 (pUC 9), as described for the SHA assay, were tested for
removal of hemagglutination inhibition activity of anti-B. gingivalis
antiserum. Since it is necessary to determine the minimum number of B.
gingivalis cells which produces hemagglutination before performing the
hemagglutination inhibition assay, a direct hemagglutination assay of
antigen-expressing clones together with B. gingivalis was first
performed.
The direct hemagglutination assay of these clones demonstrated
that clones 2, 5, and 7 did agglutinate sheep erythrocytes, whereas E.
coli JM 109 (pUC 9) did not (Figure 9). The hemagglutination titer of
clone 2 was 2 and that of clones 5 and 7 agglutinated erythrocytes at
the undiluted suspension. In addition, clone 5 was found to auto-
agglutinate when resuspended in PBS, pH 7.2.
Restriction Maps
Since three of the antigen-expressing clones were found to
agglutinate erythrocytes, the possibility arose that they may have
common DNA inserts which encode the same function. Clone 2 resulted
from some kind of DNA rearrangement, i.e., clone 2 DNA when cut by Hind
III, did not result in pUC 9 and insert bands but showed one large band
and one small band as described in Chapter Two (Figure 2, lane 11).
The rearrangement may have resulted from a deletion or other
rearrangement at one Hind III end of the insert and another Hind III
end may still be intact. In order to obtain information as to the
nature of the rearrangement of clone 2 and the relationship of the
three hemagglutinating clones to one another, restriction maps of these
three clones were generated.


Figure 9. Hemagglutination of sheep erythrocytes. Bacterial suspensions were
diluted in 2 fold serial dilutions and mixed with an equal volume of 0.2% (V/V)
erythrocytes as described in the methods. Row 1 is the undiluted bacterial suspensions.
(A) B. gingivalis 381, (B) E. ooli JM 109 (pUC 9), (C) clone 2, (D) Clone 3,
(E) Clone 4, (F) Clone 5, (G) Clone 7.


59


60
The recombinant plasmids of clones 2, 5, and 7 were restricted
with several restriction endonucleases and analyzed in 1.2% agarose
gels as shown in Figures 10, 11, arid 12. A restriction map of each
clone was generated as shown in Figures 13, 14, and 15. A schematic
diagram of restriction enzyme recognition sites of these three clones
is detailed in Figure 16. This data indicates that the clone 2 insert
appears to be different from that of clones 5 and 7, whereas clones 5
and 7 have one insert fragment in common. The restriction map of clone
2 revealed that the Hind III site of the DNA insert at the amino
terminal end of the 3-galactosidase gene was still intact but a
deletion occurred at the other end of the insert and included most of
the linker. The linker region with recognition sites of Pst I, Sal I,
Bam HI and Sma I was deleted but the Eco RI site was still intact as
wrell as other sites upstream such as Pvu II and Nar I.
Southern Blot Analysis
To further confirm the results of the restriction maps,
32P-labeled clone 7 recombinant DNA was used as a probe for
hybridization of restricted recombinant plasmids by Southern blot
analysis. Clone 2 DNA restricted with Hind III, Eco RI, and Sma I
resulted in DNA fragments of pUC 9 and 4 pieces of insert of
approximately 1,400, 1,300, 420, and 150 bp (Figure 17, panel A, lane
2). Clone 5 DNA restricted with Hind III resulted in fragments of pUC
9 and 2 pieces of insert of approximately 4,800 and 760 bp (Figure 17,
panel A, lane 3). Fragment bands of pUC 9 and inserts of approximately
2,800, 2,000, and 760 bp were generated from digestion of clone 5 DNA
with Hind III and Bam HI (Figure 17, panel A, lane 5). Clone 7 DNA


Figure 10. Agarose gel electrophoresis of restriction digests of the recombinant
plasmid from clone 2.
Lanes: 1, DNA marker-Hind III digest of lambda DNA; 2, pUC 9 digested with Hind III;
3, pUC 9 digested with Pvu II; and recombinant plasmid from clone 2 digested with;
4, Hind III; 5, Pvu II; 6, Pvu II and Hind III; 7, Eco RI; 8, Eco RI and Hind III;
9, Sma I; 10, Sma I and Hind III; 11, Sma I and Eco RI; 12, Eco RI and Pvu II; 13,
Nar I; 14, Eco RI and Nar I; 15, Bam HI; 16, Hind III and Bam HI; 17, Sma I and
Bam HI; 18, Ca I; 19, Ca I and Hind III; 20, Eco RI and Ca I.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
23.1
9.4
6.5
4.3
2.3
2.0
0.5
i ^
**spp
# "* ,
y
yMt£
u -.i
*
i
. w
1
1
1
1
I
W
V ^
M M **
, *
M '
4 INI tfca# (Ml
u
.
*
^4.
' .
1 i M U mu
li M ^
. zSf' jfr .-
M
*
r 9>'
'v ; $. -.*
* * 4 .

W4t
.-
'<' ?vV ,v\
-.fe
1.

CT>
PO


Figure 11. Agarose gel electrophoresis of restriction digests of the recombinant
plasmid from clone 5.
Lanes: 1, DNA marker-Hind III digest of lambda DNA; 2, pUC 9 digested with Hind III
and recombinant plasmid from clone 5 digested with; 3, Hind III; 4, Bam HI; 5, Hind
III and Bam HI; 6, Sal I; 7, Hind III and Sal I; 8, Hind III and Eco RV; 9, Stu I;
10, Hind III and Stu I; 11, Stu I and Sal I; 12, Stu I and Eco RV; 13, Stu I and
Asp 718; 14, Hind III and Asp 718; 15, Bam HI and Asp 718; 16, Bam HI and Stu I;
17, Eco RI; 18, Hind III and Eco RI.


23.1
9.4
6.5
4.3
2.3
2.0
1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 18
CTl
-P*
0.5


Figure 12. Agarose gel electrophoresis of restriction digests of recombinant
plasmids from clones 5 and 7.
Lanes: 1, DMA marker-Hind III digest of lambda DNA; 2, pUC 9 digested with Hind III;
recombinant plasmids from clones 5 and 7 in adjacent lanes digested with; 3 and 4,
Hind III; 5 and 6, Hind III and Bam HI; 7 and 8, Hind III and Asp 718; 9 and 10,
Hind III and Stu I; 11 and 12, Sal I; 13 and 14, Hind III and Eco RI.


23.1
9.4
6.5
4.3
2.3
2.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
CT>
CTi


Figure 13. REstriction map of the recombinant plasmid from
clone 2. The heavy line represents B. gingivalis DNA insert.


Pvu II
Hind III
Bam HI
Hind HI
Bam HI
Ca I
Sma I
Nar I
Ca I
Sma I
Nar I
EcoRI
co
fO
b
o
o
o
o
o
o
cr
X)
cr^
00


Figure 14. Restriction map of the recombinant plasmid from
clone 5. The heavy line represents B. gingivalis DMA insert.


Hind III
Eco RV
Sal I
Stu I
Hind III
Bam HI
Asp 718
Stu I
Eco Rl
Hind III
Pst I
Sal I
Bam HI
Sma I
Eco Rl
o
o
o
i o
cr
o
o


Figure 15. Restriction map of the recombinant plasmid from
clone 7. The heavy line represents B. aingivalis DNA insert.


Hind III
Pst I
Sal I
Bam HI
Sma I
Eco Rl
cr
o
o
4.800 4,000 3,000 2,000 1.000


Figure 16. Schematic diagram of restriction enzyme recognition sites of recombinant
plasmids from clones 2, 5, and 7. The solid lines represent B. gingivalis DNA
inserts. The hatched boxes represent pUC 9 regions.


/ 3NO10 Z//////YAi in YA'///////.
Hina,
'0 ?
ia
<>/
a,
*0
<>/
Ca I
Sma I
k- Mari<3-
H
K
*>!
Eco FU
Hind III
Pst I
Sal I
Bam H I
Sma I
Eco R I
Hind III
Pst I
Sal I
Bam H I
Sma I
Eco R I
O
l
o
z
m
ro
O
r~
O
z
m
cn
U


Figure 17. Southern blot analysis of the hemagglutinating E. ooli
(A) Agarose gel (1.2%) showing restriction digests of the DNA.
Lanes: 1, pUC 9 digested with Hind III; 2, recombinant plasmid
from clone 2 digested with Hind III, Eco RI, and Sma I; 3, recom
binant plasmid from clone 5 digested with Hind III; 4, recombinant
plasmid from clone 7 digested with Hind III; 5, recombinant plasmid
from clone 5 digested with Hind III and Bam HI; 6, recombinant
plasmid from clone 7 digested with Hind III and Bam HI.
(B) Autoradiograph of DNA in panel A after Southern transfer and
hybridization with 32p_iabeled recombinant DNA from clone 7.


76


77
restricted with Hind III alone and Hind III together w'ith Bam HI
resulted in pUC 9 and an insert of 4,800 bp (Figure 17, panel A, lane
4), and pUC 9, insert of 2,800 and 2,000 bp (Figure 17, panel A, lane
6), respectively.
Hybridization of these transferred restricted DNAs demonstrated
that the clone 7 probe hybridized to pUC 9 and the common insert of
clones 5 and 7 but not to the insert of clone 2 (Figure 17, panel B).
Subcloning of Clone 5 for Autoagglutination and Hemagglutination
Clone 5 was found to agglutinate erythrocytes and autoagglutinate
while clone 7 was only able to agglutinate erythrocytes. Clone 5 has
an insert of 760 bp in addition to the common insert of 4,800 bp of
clone 7. This data suggested that the 760 bp insert might encode for
the autoagglutinating activity and the 4,800 bp fragment for the
hemagglutinating activity of clone 5. The recombinant plasmid of clone
5 was thus digested with Hind III to generate pUC 9 and inserts of
4,800 and 760 bp. Each insert band was isolated from the agarose gel
and ligated to Hind III cut pUC 9 and transformed into E. coli JM
109. The plasmids w'ere isolated from these transformants and digested
with restriction endonucleases. Subclones with different orientations
of the insert were obtained. Subclones of 760 bp inserts w'ere
designated clone 5.1 and 5.2 and the subclones of 4,800 bp inserts,
clone 5.3 and 5.4. Recombinant plasmids of clones 5.1 and 5.2 digested
wdth Hind III did result in pUC 9 and the 760 bp inserts (Figure 18,
lanes 2 and 3), and different patterns of restricted DNAs were seen
when digested with Sal I (Figure 18, lanes 6 and 7). Hind Ill-
restricted recombinant plasmids of clones 5.3 and 5.4 revealed pUC 9


78
and inserts of 4,800 bp (Figure 18, ianes 4 and 5), while Eco RI-
restricted recombinant plasmids showed different patterns (Figure 18,
lanes 8 and 9). Both clones 5.1 and 5.2 were able to autoagglutinate
when resuspended in PBS, pH 7.2, but could not agglutinate
erythrocytes. Clones 5.3 and 5.4 were both able to agglutinate
erythrocytes but did not autoagglutinate.
Western Blot Analysis of B. gingivalis Antigens Synthesized in
hemagglutinable E. coli
Upon Western blot analysis of clone 2, a protein antigen of
approximately 125 K and a smear of lower molecular weight were detected
using E. coli adsorbed anti-J3. gingivalis antiserum but antigens
expressed in clones 5 and 7 were not detected by Western blot
analysis (described in Chapter Two). In an attempt to detect antigen
expression of DNA inserts in clones 5 and 7 and to achieve expression
of a more stable product from clone 2, the recombinant plasmids from
these clones were transformed into E. coli LC 137 lhtpR(AmTs) lonR 9
(Ts) laciAm) trp{Am) pho(Am) rpsL supCiTs) mal(Am) tsx::TnlO] kindly
provided by A. L. Goldberg. This bacterial strain has mutations in the
htpR and Ion genes, the products of which are involved in intracellular
protein degradation. However, this attempt was not successful since
the expressed antigen of clone 2 was still degraded and antigen
expression of clones 5 and 7 was not detected.
Identification of Native B. gingivalis Antigens
In order to determine the native B. gingivalis antigens which
clone 2 expressed, antisera against clone 2 were made in rabbits for


Figure 18. Agarose gel electrophoresis of recombinant plasmids
from clones 5.1, 5.2, 5.3, and 5.4.
Lanes: 1, DNA marker-Hind III digest of lambda DNA; 2-5,
recombinant plasmids from clones 5.1, 5.2, 5.3, and 5.4 digested
with Hind III; 6 and 7, recombinant plasmids from clones 5.1 and
5.2 digested with Sal I; 8 and 9, recombinant plasmids from clones
5.3 and 5.4 digested with Eco RI.


80


81
use as a probe in Western blot analysis. Pooled anti-clone 2 antiserum
had a titer of 1:16,000 against B. gingivalis whole cell antigen. This
antiserum was adsorbed exhaustively with E. coli JM 109 (pUC 9) until
the anti-FL coli titer was reduced from 1:50,000 to 1:10 in the E. coli
whole cell ELISA. The adsorbed antiserum, diluted to 1:200, was used
as a probe to detect antigens separated in a 12.5% SDS polyacrylamide
gel and transferred to a nitrocellulose sheet. As can be seen in
Figure 19, this antiserum reacted with 2 major bands of approximately
MWs 43,000 and 38,000 and 2 bands of MWs 32,000 and 30,000 in B.
gingivalis cell lysate antigen and the 125 K protein band of expressed
antigen in clone 2. Normal rabbit serum reacted to a common 40,000
molecular weight band of all the clones and E. coli JM 109 (pUC 9).
In order to prove that the B. gingivalis reactive polypeptides are
exclusively B. gingivalis proteins, the native B. gingivalis antigens
were reacted to E. coli adsorbed anti-clone 2 antiserum, B. gingivalis
cell lysate antigen and clone 2 whole cell antigen were again separated
in 12.5% SDS polyacrylamide gel. Upon transfer to a nitrocellulose
sheet, each was reacted with 1) E. coli adsorbed anti-clone 2 antiserum,
2) B. gingivalis adsorbed anti-clone 2 antiserum, and 3) antisera to
E. coli JM 109 harboring pUC 9 with an Eikenella corrodens DNA insert.
As can be seen in Figure 20, E. coli adsorbed anti-clone 2 reacted to B.
gingivalis cell lysate at 2 major bands of MWs 43,000 and 33,000, 2
bands of MWs 32,000 and 30,000 and 3 faint bands of higher molecular
weight of approximately 110,000, 90,000, and 75,000 daltons. This
adsorbed antiserum also reacted to a 125,000 MW band of expressed
antigen in clone 2. B. gingivalis adsorbed anti-clone 2 and anti-
E. coli JM 109 harboring pUC 9 with Eikenella DNA insert antisera did


Figure 19. Western blot analysis of native B. gingivalis antigens expressed by clone 2.
Lanes: 1, B. gingivalis cell lysate (40 pg); 2 to 7, whole cell samples of clones 2, 3,
4, 5, 7, and E. aoli JM 109 (pUC 9) as described in the methods.
(A) The blot was probed with E. coli-adsorbed anti-clone 2 antiserum.
(B) The blot was probed with normal rabbit serum.


83


Figure 20. Western blot analysis of native B. gingivalis antigens
expressed by clone 2.
Lanes: 1, B. gingivalis cell lysate (40 ug); 2, whole cell sample
of clone 2.
(A) The blot was probed with E. coli adsorbed anti-clone 2 antiserum.
(B) The blot was probed with B. gingivalis adsorbed anti-clone 2
anti serum.
(C) The blot was probed with antiserum against E. coli JM 109
harboring pUC 9 with an Eikenella DNA insert.


85
A
94 K
67K
43K
30K.
20K.
14 K
B
C
1 2
$**$?.**
fir*'


86
not react to B. gingivalis antigens or to the expressed antigen o.
clone 2 but reacted with E. coli antigens in clone 2.
To define the native B. gingivalis antigens which clones 5 and 7
expressed, antisera against clones 5 and 7 were also made in rabbits
and had titers of 1:800 and 1:1,600 to B. gingivalis antigens. These
antisera exhaustively adsorbed with E. coli were used to identify the
reactive native B. gingivalis antigens. Antisera against clones 5 and 7
at the dilution of 1:5 and 1:10 were found to react with 2 bands of
approximately 43,000 and 38,000 daltons in B. gingivalis cell lysate
antigen preparation but did not react to the expressed clone 2 antigen
(Figure 21). This antiserum also reacted to a common band of
approximately 36,000 daltons of E. coli antigen in each clone and E.
coli JM 109 (pUC 9). Normal rabbit serum did not react to any B.
gingivalis antigens (Figure 21).
In order to determine if the anti-clones 2, 5, and 7 antisera were
reacting with the same B. gingivalis polypeptides or with different
peptides of similar migration rates, four samples of B. gingivalis
cell lysate antigens were separated in a 12.5% SDS polyacrylamide gel,
transferred to nitrocellulose paper and reacted with anti-clone 2
antiserum diluted 1:200, anti-clone 5 antiserum diluted 1:5, anti-clone
7 antiserum diluted 1:10, and a mixture of anti-clones 2, 5, arid 7
antisera at final concentrations of 1:200, 1:5, and 1:10, respectively.
Any differences in the pattern of reaction were undiscernable in these
4 blots (Figure 22).


Figure 21. Western blot analysis of native B. gingivalis antigen expressed by clone 7.
Lanes: 1, B. gingivalis cell lysate (40 ug); 2 to 5, whole cell samples of clones 2, 5,
7, and E. coli JM 109 (pUC 9) as described in the methods.
(A) The blot was probed with E. coli-adsorbed anti-clone 7 antiserum.
(B) The blot was probed with normal rabbit serum.


A
1 2 3 4 5
94 K
67K .
43K
30K
20 K
14 K
vM::
|
-R >*
B
1 2 3 4 5
oo
oo


Figure 22. Western blot analysis of native B. gingivalis
antigens expressed by clones 2, 5, and 7. Forty ug of
B. gingivalis cell lysate was separated in a 12.5% SDS
polyacrylamide gel, transferred to a nitrocellulose sheet,
and probed with 1) E. coli adsorbed anti-clone 2 antiserum
diluted 1:200; 2) E. coli adsorbed anti-clone 5 antiserum
diluted 1:5, 3) E. coli adsorbed anti-clone 7 antiserum
diluted 1:10; and 4) a mixture of the above antisera.


90
12 3 4
__ ]
94 K
67 K
43K
30K
20 K
14 K


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ER8XORGYJ_2IUIY5 INGEST_TIME 2012-02-20T21:16:43Z PACKAGE AA00009090_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


MOLECULAR CLONING AND CHARACTERIZATION OF
PACTEROIDF.S GINGIVAL1S ANTIGENS
BY
SOMYING TUMWASORN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOO
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988

ACKNOWLEDGEMENTS
I would like to express my appreciation to my advisor, Dr. Ann
Progulske for her guidance, friendship, and assistance in this study
and in the preparation of this dissertation. I have enjoyed working
with her and realize that I truly like tills kind of research.
I really appreciate Dr. Donna H. Duckworth, my committee member,
for her guidance and technical assistance in the area of molecular
genetics and for her moral support and friendship since the beginning
of my graduate study.
Special thanks go to my committee members, Dr. Clay B. Walker, Dr.
Wülliam B. Clark, Dr. William P. McArthur, Dr. Anthony F. Barbet, my
external examiner, Dr. Francis L. Macrina, and the chairman of the
Department of Immunology and Medical Microbiology, Dr. Richard R.
Moyer, for their constructive criticisms and technical assistance in
this study.
I also thank the Fulbright Foundation for providing financial
support for the beginning of my study and the Thai Government for
granting me a leave of absence.
I would also like to thank the fellow' graduate students and
scientists, Dr. Connie D. Young, and Dr. Thomas A. Brown for their
technical assistance, discussions and friendship.
I wish to express my deepest gratitude to my parents Sanguan and
Chaufa Juijaitrong who provided me wdth the educational background and

encouragement, their love, concern, and devotion.
Finally, I wish to extend my appreciation to my husband Sornthep
and sons, Pattarawuth and Nattapol, for their love, patience,
encouragement and dedication that created the necessary environment to
permit the conclusion of this work.

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT viii
CHAPTER
ONE INTRODUCTION 1
Bacteroides gingivalis as the Periodontopathogen 1
Pathogenicity of B. gingivalis 2
Application of Recombinant DNA Techniques to
the Study of Periodontal Disease 9
TWO CLONING AND EXPRESSION OF BACTEROIDES GINGIVALIS
ANTIGENS IN ESCHERICHIA COLI 12
Introduction 12
Materials and Methods 14
Results 24
Discussion 42
THREE CHARACTERIZATION OF BACTEROIDES GINGIVALIS ANTIGENS
SYNTHESIZED IN ESCHERICHIA COLI 48
Introduction 48
Materials and Methods 50
Results 55
Discussion 97
FOUR CONCLUSION 103
LITERATURE CITED 108
BIOGRAPHICAL SKETCH 118
iv

LIST OF TABLES
Table Page
1 Characterization of E. coli transformants which
express B. gingival is antigens 26
2 Titer of anti-fb gingivalis against E. coli
transformants which express B. gingivalis antigens 39
3 Inhibition of adherence to SHA by adsorbed anti-fi.
gingivalis antisera 56
4 Inhibition of hernagglutinating activity of B.
gingivalis by anti-hemagglutinating E. coli
antisera 96
5 Inhibition of hernagglutinating activity of B.
gingivalis by adsorbed anti-fb gingivalis
antiserum 98
v

LIST OF FIGURES
Figure Page
1 Map of pUC 9 16
2 Agarose gel electrophoresis of recombinant plasmids 28
3 Agarose gel electrophoresis of different restriction
digests of recombinant plasmid from clone 3 31
4 Agarose gel electrophoresis of different restriction
digests of recombinant plasmids from clones 5, 6, 7,
and 8 33
5 Agarose gel electrophoresis of different restriction
digests of recombinant plasmids from clones 1, 2,
and 4 35
6 Hybridization of recombinant plasmids with 3ZP labeled
B. gingivalis DNA probe 38
7 SDS-PAGE (on 12.5% acrylamide) and Western blot analysis
of expressed B. gingivalis antigens 41
8 SDS-PAGE (on 5% acrylamide) of expressed B. gingivalis
antigen in clone 2 44
9 Hemagglutination of sheep erythrocytes 59
10 Agarose gel electrophoresis of restriction digests of
the recombinant plasmid from clone 2 62
11 Agarose gel electrophoresis of restriction digests of
the recombinant plasmid from clone 5 64
12 Agarose gel electrophoresis of restriction digests of
recombinant plasmids from clones 5 and 7 66
13 Restriction map of the recombinant plasmid from
clone 2 68
14 Restriction map of the recombinant plasmid from
clone 5 70
15 Restriction map of the recombinant plasmid from
clone 7 72
16 Schematic diagram of restriction enzyme recognition
sites of recombinant plasmids from clones 2, 5,
and 7 74
17 Southern blot analysis of the hemagglutinating
E. coli 76
vi

Figure Page
18 Agarose gel electrophoresis of recombinant plasmids
from clones 5.1, 5.2, 5.3, and 5.4 80
19 Western blot analysis of native B. gingivalis
antigens expressed by clone 2 83
20 Western blot analysis of native B. gingivalis
antigens expresses by clone 2 85
21 Western blot analysis of native B. gingivalis
antigen expressed by clone 7 88
22 Western blot analysis of native B. gingivalis
antigens expressed by clones 2, 5, and 7 90
23 Detection of B. gingivalis antigens synthesized
by clones 2, 5, and 7 as determined by Western blot
analysis 93
24 ELISA of anti-clone 2 antiserum adsorbed with
various numbers of cells 95

Abstract of a Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MOLECULAR CLONING AND CHARACTERIZATION OF
BACTEROWES GINGIVALIS ANTIGENS
By
Somying Tumwasorn
April 1988
Chairman: Ann Progulske
Major Department: Immunology and Medical Microbiology
Bacteroides gingivalis, a Gram-negative anaerobic bacterium, is
strongly implicated as an etiological agent of periodontal disease.
However, its exact role in the disease process has not yet been
established. Recombinant DNA technology w-as applied as an initial
approach to a molecular study of B. gingivalis antigens by preparing
genomic libraries of B. gingivalis strain 381 in E. coli JM 109 via a
pUC 9 plasmid vector. Detection of the expression of B. gingivalis
antigens wras achieved by using E. coli adsorbed rabbit anti-5.
gingivalis sera.
Five different clones w-ere found to stably exhibit B. gingivalis
antigen expression. Characterization of the antigen-expressing clones
demonstrated that clones 2, 5, and 7 agglutinate sheep erythrocytes
whereas E. coli JM 109 (pUC 9) does not. Clones 5 and 7 w'ere found to
have one insert fragment in common and this insert was found to have
little or no homology to the insert of clone 2. Clone 5 is also able
vi i i

to autoagglutinate and it was found that a 760 bp DNA fragment codes
for this activity. The common insert of clones 5 and 7 appears to
have a Bacteroides promoter and to code for the hemagglutinating
activity of these clones. The clone 2 insert does not have a
Bacteroides promoter and is under the control of plasmid lac promoter.
Antisera against clones 2, 5, and 7 were found to inhibit the
hemagglutinating activity of B. gingivalis whereas adsorption of
anti-5, gingivalis antiserum with clones 2, 5, and 7 partially removed
the hemagglutination inhibition activity. However, these clones do not
remove the saliva-treated hydroxyapatite (SHA) adherence inhibition
activity of anti-5, gingivalis antiserum. Western blot analysis of
5. gingivalis cell lysate antigens using E. coli adsorbed antisera
against clones 2, 5, and 7 demonstrated that all antisera reacted to 2
major bands of MWs 43,000 and 38,000, which have been reported to be
the major bands of the 5. gingivalis hemagglutinin. E. coli
adsorbed anti-clones 5 and 7 antisera did not react to the 125,000
protein band expressed in clone 2. In addition, adsorption assays
demonstrated that the epitope of the expressed antigen in clone 2 is
not related to that of clones 5 and 7. A number of experiments are
proposed to further characterize the 5. gingivalis hemagglutinin genes,
the nat:ve hemagglutinin molecules, and the significance of
hemagglutinins in periodontal disease.
IX

CHAPTER ONE
INTRODUCTION
Periodontal disease (PD) is a chronic inflammatory disease which
results in the destruction of the supporting tissues of teeth (Kagan,
1980). Although the specific microbial etiology of PD is not known, it
is widely accepted that bacteria are the contributing agents of the
disease for the following reasons (Socransky, 1977): 1) disease
correlates with the presence of plaque, 2) antibiotics are effective in
treatment of PD, and 3) implantation of certain genera of bacteria into
gnotobiotic rats results in PD of infected but not of control rats.
Bacteroides gingivaiis as the Periodontopathogen
The presence of a complex microflora in the subgingival crevice
has complicated the identification of the specific etiologic agents of
PI). However, several studies (Socransky, 1977; Slots, 1979; White ai d
Mayrand, 1981) indicate that a few genera, primarily Gram-negative
anaerobes, appear to be associated with disease progression. For
example, the proportion of Gram-negative anaerobes, especially black-
pigmented Bacteroides, increases markedly in the subgingival flora with
increasing severity of PD. Bacteroides gingivaiis, previously oral
Bacteroides asaccharolyticus (Coykendall et al., 1980), is the black-
pigmented Bacteroides which has emerged as a key putative
periodontopathogen for a number of compelling reasons.
1

2
B. gingivalis is the predominant bacterial species isolated from
periodontal lesions of patients with severe adult periodontitis
(Slots,1977; Tanner et al., 1977). Patients with adult periodontitis
have been found to have higher levels of IgG antibodies to B. gingivalis
than normal adults (Mouton et. al., 1981) and local immunity to B.
gingivalis is greater in the more advanced cases than in the early
forms of PD (Kagan, 1980). Serum antibody titers to B. gingivalis have
been reported to decrease after therapy of adult periodontitis
patients, suggesting that antibodies to B. gingivalis result from
infection of this organism (Tolo et al., 1982). B. gingivalis is also
the most interesting and potentially virulent bacterium cultivable from
the subgingival crevice with respect to its capacity for breakdown of
tissues and host defense mechanisms (Mayrand and McBride, 1980; Van
Steenbergen et al., 1982; Nilsson et al., 1985). In addition, B.
gingivalis appears to be a causative agent of experimental
periodontitis in animals. When B. gingivalis is implanted as the
monocontaminant in gnotobiotic rats, it causes accelerated alveolar
bone loss (CrawTord et al., 1977). In a longitudinal study of alveolar
bone loss in Macaca arctoides (Slots and Hausmann, 1979), the proportion
of B. gingivalis-type isolates reportedly increased from a minority of
the cultivable microbiota prior to bone loss to a majority of the
microflora when alveolar bone loss was detectable.
Pathogenicity of B. gingivalis
Although B. gingivalis has been strongly implicated as an
etiological agent of adult periodontitis, its exact role in the disease
process has not yet been established. In order to produce PD, it is

3
likely that bacteria and/or their products may lead to the destruction
of the gingival tissues by direct action or indirectly by eliciting an
immune response which is detrimental to the host tissues.
Periodontopathic bacteria such as B. gingivalis must possess
characteristics which enable them to colonize the host, survive in the
periodontal pocket, possibly invade the gingival tissues, and to
destroy the collagenous periodontal ligament, the alveolar bone, and
other tissue components surrounding the tooth (Slots and Genco, 1984).
Colonization
It is now recognized that colonization of the oral cavity and many
other mucosal environments requires the adherence of bacteria to the
surface in order to resist the cleansing action of glandular secretions
(Gibbons and Van Houte, 1975; 1980). The adherence of bacteria to host
tissues is thus a prerequisite for colonization, which is the initial
event in the pathogenesis of disease (Gibbons and Van Houte, 1975).
The mechanisms of bacterial adherence involve both ionic and other
physical (covalent) forces. Many, if not all pathogenic bacteria
possess specific ligands on their surfaces, called "adhesins" which
bind to complementary components on host tissues (Gibbons and Van
Houte, 1980). The mechanisms of adherence may involve the interaction
of carbohydrate binding proteins, or lectins, on bacterial surfaces
with carbohydrate-containing receptors on host cells. Binding
properties of adhesins may also be facilitated by their hydrophobic
domains (Gibbons, 1984).

4
Components of bacteria which mediate attachment to host tissues
include surface structures such as fimbriae, capsular materials,
lipopolysaccharides, and membrane-associated extracellular vesicles
(Slots and Genco, 1984). In the oral cavity, bacteria can attach to
host tissues as well as Gram-positive bacteria in pre-formed plaque
(Slots and Gibbons, 1978). The nature of the binding sites on teeth
and oral tissues to which Gram-negative bacteria attach has not been
well established. In vitro, B. gingivalis can attach to and agglutinate
erythrocytes (Okuda and Takazoe, 1974; Slots and Gibbons, 1'. 78; Slots
and Genco, 1979; Okuda et al., 1981), can adhere in high numbers to
human buccal epithelial cells (Slots and Gibbons, 1978; Okuda et al.,
1981), crevicular epithelial cells derived from periodontal pockets
(Slots and Gibbons, 1978), and surfaces of Gram positive bacteria
present, in plaque, (Slots and Gibbons, 1978; Schwarz et al., 1987). B.
gingivalis is also able to adhere to untreated and saliva-treated
hydroxyapatite, but in comparatively low numbers (Slots and Gibbons,
1978). B. gingivalis has also been reported to bind to HR9 matrix, a
material similar to the basement membrane barrier underlying connective
tissue (Leong et al., 1985). Recently, it has been reported that B.
gingivalis can bind fibrinogen and possibly colonize host tissues by
attaching to fibrinogen-coated surfaces (Lantz et al., 1986).
Bacterial antagonism may also play an important role in mediating
the colonization of B. gingivalis. In normal adults, Streptococcus
sanguis is a predominant organism in supra- and subgingival plaque.
S. sanguis elaborates sanguicin, a bacteriocin which in vitro inhibits
black-pigmented Bacteriodes (Nakamura et al., 1981). Experimental
studies in humans have shown that the number of Streptococcus species,

5
including 5. sanguis, are decreased, while those of Actinomyces and
black pigmented Bacreriodes are increased (Leosche and Syed, 1978) in
gingivitis. The mechanism of the proportional decrease of 5. sanguis
is not. known but this shift seems to be one of the triggers for the
initiation of compositional changes in the subgingival flora. A
decrease of sanguicin production may permit the growth of Actinomyces
species and black pigmented Bacteriodes (Takazoe et al., 1984). The
growth of B. gingivaiis may be enhanced by herriin when bleeding occurs
in gingivitis, since hemiri is a required factor for the cultivation of
B. gingivaiis. Recently, it has been reported that B. gingivaiis grown
under hemin-limited conditions has a reduced virulence in mice compared
with bacteria cultured in an excess of hemin (McKee et al., 1986).
When colonization of B. gingivaiis occurs, there seems to be a
change in the bacterial composition in the periodontal pocket. This
could be explained by studies of Nakamura et al (1978; 1980) which have
demonstrated that B. gingivaiis produces the black pigment hematin
which inhibits the growth of some Gram-positive bacteria, including S.
mutans, S. mitis, A. viscosus, A. naeslundii, A. israelii, Bacterionema
matruchotii, Corynebacterium parvum and Propionibacterium acnés.
Factors other than inhibitory substances could also affect the
colonization of B. gingivaiis, i.e., the nature of specific antibody
and other components in gingival fluid as well as the interactions
between the new predominant colonizer and other pre-existing
residents (Takazoe et al., 1984).

6
Evasion of Host Defense
B. glngivalis may survive in the periodontal pocket because it
resists phagocytosis. Sundqvist et al. (1982) demonstrated that in
vitro, most strains of B. gingivalis exhibit a higher resistance to
phagocytosis than do less pathogenic strains and that impaired
phagocytosis of this bacterial species is related to capsular material.
The Bacteroides capsule only poorly activates complement, therefore it
may function to decrease PMN chemotactic stimulus by masking LPS which
strongly activates complement (Okuda et al., 1978). Various
experiments have verified that the black pigmented Bacteroides strains
do not stimulate a strong PMN chemotactic response (Sveen, 1977 a,b;
Lindhe and Socransky, 1979; Sundqvist and Johansson, 1980).
Most strains of B. gingivalis demonstrate resistance to serum
bactericidal systems (Sundqvist and Johansson, 1982). B. gingivalis
has also been shown to degrade the plasma proteins which are important
in the host defense, such as the complement factors C3 and C5
(Sundqvist et al., 1985), immunoglobulins G, A, and M (Kilian, 1981;
Sundqvist et al., 1985), alpha-l-proteinase inhibitor,
alpha-2-macroglobulin (Carlsson et al., 1984a), haptoglobin, and
hemopexin (Carlsson et al., 1984b). It has also been shown that
B. gingivalis has the capacity to inactivate and degrade the plasma
proteins of importance in the initiation and control of the
inflammatory response such as Cl- inhibitor, antithrombin, and aipha-2-
antiplasmin (Nilsson et al., 1985). In addition, B. gingivalis can
degrade fibrinogen (Lantz et al., 1986) and fibrin (Mayrand and
McBride, 1980; Wikstrom et al., 1983); therefore, no effective fibrin
barrier is formed around the organism. B. gingivalis thus appears to

7
be an organism fully capable of inactivating the host defense
mechanisms against invading bacteria.
Periodontal Tissue Destruction
B. gingivalis possesses a number of components with the potential
to destroy gingival tissue constituents as follows: The B. gingivalis
lipopolysaccharide possesses strong bone resorptive activity (Nair et
al., 1982), and inhibits the growth of cultured fibroblasts derived
from healthy and periodontally diseased human gingiva (Layman and
Diedrich, 1987). The lipopolysaccharide is also a suspected component
that stimulates mononuclear cells to produce a factor which strongly
stimulates osteoclast-mediated mineral resorption (Bom-Van Noorloos et
al., 1986). B. gingivalis proteolytic enzymes, especially collagenase
(Mayrand and McBride, 1980; Robertson et al., 1982; Mayrand and
Grenier, 1985) and a trypsin-like protease (Slots, 1981; Laughon et
al., 1982) may be directly involved in periodontal tissue destruction.
Enzymes other than proteases may also play an important role in the
pathogenesis of periodontal disease. For example, alkaline and acid
phosphatases (Slots, 1981; Laughon et al., 1982) may cause alveolar
bone breakdown since it has been shown that bacterial phosphatases
could cause alveolar bone breakdown (Frank and Voegel, 1978).
Bacterial products, i.e., butyrate, propionate (Singer and Buckner,
1981) and volatile sulfur compounds (Tonzetich and McBride, 1981) are
also suspected to be toxic to periodontal tissues. Recently, it has
been reported that B. gingivalis possesses a cartilage-degrading ability
which is suspected to be due to its ability to degrade proteinase
inhibitors (Klamfeldt, 1986).

8
It has been suggested that periodontal tissue destruction is
mediated not only by bacteria and their products but aiso by the host
defense mechanisms (Horton et ah, 1974; Nisengard, 1977). For
example, cell mediated immunity has been shown to correlate with the
periodontal status of patients (Ivanyi and Lehner, 1970; Patters et
al., 1976; Patters et al., 1979). B. gingivalis was found to stimulate
significantly more lymphoproliferative response iri patients with
destructive periodontitis than those of normal subjects or those with
gingivitis (Patters et al., 1980). A lymphoproliferative response
results in the production of lymphokines, several of which can account
for some of the destructive effects in periodontal disease. For
example, alpha-lymphotoxin can cause cell death and osteoclast
activating factor can stimulate osteoclastic bone resorption (Horton et
al., 1972). Macrophages have also been suggested to play a role in
periodontal tissue destruction. Macrophages may be stimulated by
bacterial antigens such as LPS (Wahl, 1982), or by lymphokines (Mooney
and Waksman, 1970; Wahl et al., 1975), and subsequently produce
tissue-degrading enzymes such as collagenase and other proteases (Wahl
et al., 1975; Wahl, 1982). Recently, it has been demonstrated that
lipopolysaccharide of B. gingivalis can induce circulating mononuclear
cells to release collagenase-inducing cytokines. The cytokines then
induce collagenase synthesis in human gingival fibroblasts (Health et
al., 1987). In addition, IgE, mast cells and basophils may also play a
role in periodontal disease (Jayawardene and Goldner, 1977; Olsson-
Wennstrom et al., 1978).

9
Application of Recombinant DNA
Techniques to the Study of Periodontal Disease
The recombinant DNA techniques developed during the past few years
have proven t be powerful tools for the study of pathogenesis.
Several major antigens and virulence factors have been cloned as a
means of further characterizing their chemical natures, genetic
regulation, and function in various diseases. For example, the cloning
and expression of the Neisseria gonorrhoeae pilus protein in E.
coli (Meyer and So, 1982) has helped explain the molecular mechanism of
antigenic variation. In other studies, the cloning of several
virulence factors including exotoxins (Vodkin and Leppla, 1983; Vasil
et al., 1986; Nicosia et al., 1987), enterotoxins (Pearson and
Mekalanos, 1982), a hemolysin (Goldberg and Murphy, 1984), and a
pneumolysin (Walker et al., 1987) have allowed genetic studies of these
proteins and have facilitated the production of safer vaccines.
Cloning antigens encoded by unknown genes is made possible by preparing
a genomic library in which any gene is theoretically represented. If
the number of clones is large enough, it is hoped that any gene can be
isolated by screening the library (Perbal, 1984). Genomic libraries of
both Treponema paiiidum (Stamm et al., 1982) and Legionella pneumophila
(Engleberg et al., 1984 a;b) have been made as a first step in
isolating and characterizing their major surface antigens.
The recombinant DNA techniques have, however, been applied only
sparingly to the study of Gram-negative anaerobic pathogens and even
less to the study of the molecular mechanisms of periodontopathogenesis.
The recombinant DNA methodologies offer advantages over previous
methods used in the study of oral pathogens. Since several potential

10
periodontopathogens, including B. gingivalis, are difficult to grow to
high densities, isolation and purification of antigens, especially
those present in small amounts, are often difficult and tedious because
of a limited amount of starting material. Cloning specific structures
in an organism such as E. coli would greatly alleviate these problems
since E. coli can be grown to high densities easily and cloned structures
can be overproduced in E. coli (De Franco et al., 1981; Matsumura et al..
1986). This would facilitate the isolation and purification of that
structure or component. Also, the cloning and expression of antigens
w'ould isolate the antigens at the genetic level. The cloned antigens
can then be prepared as products devoid of other B. gingivalis antigens.
Thirdly, the cloning of B. gingivalis antigens would allow a genetic and
molecular analysis of the gene(s) which is presently difficult to do
due to the lack of a genetic system in B. gingivalis. Cloning antigens
which may be protective or have potential virulence properties is an,
as yet, relatively unexplored approach to define the role of B.
gingivalis in periodontal disease. It is an approach that may lead to
a more complete understanding of the molecular mechanisms of
periodontal disease as well as providing molecular tools for the future
production of a vaccine for periodontal disease.
The purpose of this study was to employ recombinant DNA techniques
to clone antigens of B. gingivalis as an initial step in defining their
roles in pathogenesis. The specific aims were to
1. Construct genomic libraries (clone banks) of B. gingivalis
chromosomal DNA in E. coli.
2. Identify E. coli. transformants which express B. gingivalis
antigens.

11
3. Identify cloned antigens which are potential virulence factors.

CHAPTER TWO
CLONING AND EXPRESSION OF BACTEROWES GINGIVALIS
ANTIGENS IN ESCHERICHIA COLI
Introduction
Several lines of evidence strongly implicate Bacteroides
gingivalis, a Gram-negative anaerobic bacterium, as an etiological
agent of adult periodontal disease (White and Mayrarid, 1981; Zambón et
al., 1981; Takazoe et al., 1984; Slots and Genco, 1984; Slots et al.,
1986). For example, relatively high proportions of B. gingivalis have
been isolated from adult periodontitis lesions (Slots, 1977; Tanner et
al., 1977; Spiegel et al., 1979), patients with adult periodontitis
have been found to have higher levels of IgG antibodies to B.
gingivalis than do normal adults (Mouton et al., 1981; Naito et al.,
1984), and local immunity to B. gingivalis is greater in the more
advanced cases than in the early forms of periodontal disease (Kagan,
1980). B. gingivalis also appears to be a causative agent of
experimental periodontitis in animals (Crawford et al., 1977; Slots and
Hausmann, 1979). In addition, B. gingivalis possesses a variety of
suspected virulence factors such as proteases, collagenases,
immunologlobuiin degrading enzymes, and adhesins (Slots and Genco,
1984).
Previous investigations of Bacteroides pathogenic mechanisms have
employed the isolation and purification of B. gingivalis constituents by
12

13
B. gingivaJis is the predominant bacterial species isolated from
periodontal lesions of patients with severe adult periodontitis
(Slots,1977; Tanner et al., 1977). Patients with adult periodontitis
have been found to have higher levels of IgG antibodies to B. gingivajis
than normal adults (Mouton et al., 1981) and local immunity to B.
gingivajis is greater in the more advanced cases than in the early
forms of PD (Kagan, 1980). Serum antibody titers to B. gingivalis have
been reported to decrease after therapy of adult periodontitis
patients, suggesting that antibodies to B. gingivalis result from
infection of this organism (Tolo et al., 1982). B. gingivalis is also
the most interesting and potentially virulent bacterium cultivable from
the subgingival crevice with respect to its capacity for breakdown of
tissues and host defense mechanisms (Mayrand and McBride, 1980; Van
Steenbergen et al., 1982; Nilsson et al., 1985). In addition, B.
gingivalis appears to be a causative agent of experimental
periodontitis in animals. When B. gingivalis is implanted as the
monocontaminant in gnotobiotic rats, it causes accelerated alveolar
bone loss (Crau'ford et al., 1977). In a longitudinal study of alveolar
bone loss in Macaca arctoides (Slots and Hausmann, 1979), the proportion
of B. gingivalis-type isolates reportedly increased from a minority of
the cultivable microbiota prior to bone loss to a majority of the
microflora when alveolar bone loss was detectable.
Pathogenicity of B. gingivalis
Although B. gingivalis has been strongly implicated as an
etiological agent of adult periodontitis, its exact role in the disease
process has not yet been established. In order to produce FD, it is

14
Materials and Methods
Bacteria] Strains, Plasr d and Growth Conditions
Bacteroides gingivalis 381 obtained from a stock culture was grown
on plates containing Trypticase soy agar (BBL Microbiology Systems,
Cockeysville, Md.) supplemented with sheep blood (5%), hemin (5
micrograms per ml), and menadione (5 micrograms per ml). The organism
was also grown in 10 ml of Todd-Hewitt. broth (BBL) supplemented with
heroin (5 micrograms per ml), menadione (5 micrograms per ml) and
glucose (2 milligrams per ml). Cultures were incubated in an anaerobic
O
chamber in a N2-H2-CO2 (85:10:5) atmosphere at 37 C until the log
phase of grou'th was obtained. The 10 ml broth culture was transferred
into 25 ml of the same medium and subsequently transferred to 500 ml of
O
medium. Incubation was at 37 C anaerobically until a late log phase
culture was obtained. E. coli JM 109 (rec Al, end Al, gyr A96, thi,
hsd R17 sup E44, relAl, A(lac-pro AB), CF;tra D36, proAB, lac IZ M15])
and the plasmid expression vector pUC 9 (Figure 1) were gifts of J.
Messing and have been described previously (Vieira and Messing, 1982;
Yanisch-Perron et al., 1985). E. coli JM 109 uras cultured in Luria-
Bertani (LB) medium consisting of Bacto-tryptone (10 g per liter),
Bacto-yeast extract (5 g per liter), and NaCl (5 g per liter). For
solid media. Bacto-agar uras added at a final concentration of 15 g per
liter. E. coli JM 109 transformants were selected and maintained on LB
plates containing 50 micrograms of ampicillin per ml.

Figure 1
Map of pUC 9.

16
Hind III Pst I Sal I Bam HI Sma I Eco Rl
432 424 418 412 407 402
pUC 9 (2671 base pairs)

17
Preparation oí Chromosomal DNA from B. gingivalis
Chromosomal DMA from P. gingivalis 381 was prepared by the method
of A. Das (personal communication) as follows: one to three liters of
cells were pelleted by centrifugation and washed once with lx SSC
buffer (0.87% NaC'l, 0.04% Na citrate) containing 27% sucrose and 10 mM
EDTA. The cells were pelleted and resuspended in 1/50 of the original
volume of the same buffer at 4°C. Lysozyme (5 mg/ml) in SSC was added
o
to 0.5 mg/ml, the mixture was mixed thoroughly and incubated at 37 C
for 10 minutes. Nine volumes of lx SSC containing 27% sucrose, 10 mM
EDTA and 1.11% SDS (prewarrned to 39 C) were added and the cell
O
suspension was incubated at 37 C for 10 to 30 minutes until cell lysis
was complete. In order to denature any contaminating proteins,
proteinase K was added to a final concentration of 1 mg/ml and the
lysate u'as incubated at 37° C for 4 hours. DNA was extracted twice with
phenol, twice with phenol-chloroform (1:1 by volume), and four times
with chloroform. Tu^o volumes of absolute alcohol u'ere added and the
precipitated DNA was spooled onto a glass rod. The purified DNA was
rinsed wdth 70% ethanol and suspended in TE buffer, pH 8.0 (10 mM
Tris-HCl pH 8.0, 1 mM EDTA).
Isolation of Plasmid DNA
Plasmid DNA was isolated by the method of Ish-Horowicz and Burke
(1981) in which cells wrere lysed with SDS-EDTA in the presence of NaOH.
Potassium acetate, pH 4.8, w-as added at 4°C and cell debris, protein,
RNA, and chromosomal DNA w’ere removed by centrifugation. The plasmid
wras precipitated with 2 volumes of ethanol, w’ashed with 70% ethanol,
dried, and resuspended in TE buffer at pH 7.5. The plasmid was

18
separated from contaminating RNA and any remaining chromosomal DNA by
cesium chloride density centrifugation in the presence of ethidium
bromide. Ethidium bromide and cesium chloride were removed by butanol
extraction and dialysis, respectively. The dialyzed plasmid was then
phenol- chloroform extracted, ethanol precipitated, and resuspended in
TE buffer.
Construction of Genomic Libraries
Purified B. gingivalis DNA was partially digested with Sau 3A
restriction endonuclease to create fragments of 2-10 kilobases which
were ligated to the dephosphorylated Bam HI site of vector pUC 9 with
T4 DNA ligase by standard methods (Maniatis et al., 1982). Genomic
fragments were also obtained by partial digestion of the chromosomal
DNA with Hind III restriction endonuclease and ligated to the
dephosphorylated Hind III site of pUC 9. The recombinant plasmids were
used to transform E. coli JM 109 by the method of A. Das (personal
communication). Briefly, E. coli JM 109 was grown to an early log
phase (OD550 = 0.2) in LB broth. Ten ml of the culture were
O
centrifuged at 5,000 rpm for 5 minutes at 4 C and resuspended in 2 ml
of transformation buffer 1 (TFM 1, 10 mM Tris-HCl, pH 7.5, 0.15 M
NaCl). The cells were then pelleted and resuspended in 2 ml of TFM 2
(50 mM CaCU) and incubated on ice for 45 minutes. The cells were
again pelleted and gently resuspended in 3 ml of TFM 2, and dispensed
into 0.2 ml aliquots. One tenth ml of TFM 3 (10 mM Tris-HCl, pH 7.5,
50 mM CaCl2, 10 mM MgSO varying amounts of DNA. The cells were then allowed to incubate on ice
O
for 45 minutes, and heat shocked at 37 C for 2 minutes. LB broth (0.5

19
ml) was added and the cell suspension was incubated at 07° C for 1
hour. Finally, the cells were plated on LB agar containing ampicillin
(50 micrograms per ml) and 5-brorno-4-ehloro-3-indolyJ- g-D-
galactopyranoside (X-Gal) (200 micrograms per ml) and incubated for 24
O O
to 48 hours at 37 C. All transformants were stored at -70 C in LB
broth wúth ampicillin (50 micrograms per ml) and 20% glycerol.
Preparation of Antisera
Late exponential phase cells of B. gingivalis strain 381 were
pelleted, washed with 0.01 M phosphate-buffered saline (PBS) pH 7.2,
O
and resuspended in PBS and 0.01% sodium azide at 4 C for at least 1
hour. The cells were again washed with PBS, resuspended to a
concentration of 1 x 109 cells per ml and emulsified in an equal
volume of Freund's incomplete adjuvant. The cell emulsion was injected
in 3 doses at two wreek intervals for 4 weeks subcutaneously in the back
of adult New Zealand rabbits. Each rabbit was given a booster dose 50
to 60 days later. Antisera w’ere collected from the marginal ear veins
just prior to immunization and beginning one week after the booster
O
dose. All sera were stored at -20 C.
Rabbit anti-B. gingivalis antiserum was adsorbed 4 times wúth E.
coli JM 109 harboring pUC 9 plasmid E. coii JM 109 (pUC 9) . For
each adsorption, E. coli cells from 1 liter of a stationary phase culture
O
were washed and mixed with 3 ml of serum at 4 C for 1 hour. The serum
was recovered by pelleting the cells at 5,000 x g for 20 minutes. For
sonicate adsorption, E. coli cells from 500 ml of stationary phase growth
suspended in 5 ml of PBS were disrupted by sonication and mixed with E.
O
coli cell-adsorbed serum for 1 hour at 4 C. The mixture was centrifuged

20
at 100.000 x g for 1 hour and the resulting clear serum was stored at
O
-20 C.
Assay of Antibody Titer
Sera were tested for anti-13, gingivalis and anti-E. coli activities
by an enzyme-linked immunosorbent assay (ELISA). B. gingivalis
cells suspended in carbonate-bicarbonate buffer, pH 9.6, (108 cells
O
per well) were fixed to microtiter plates at 4 C overnight. After the
wells were washed with 0.5% Tween 20 in PBS, 1% bovine serum albumin
(BSA) in PBS was added to each well, and the plates were incubated for
2 hours at room temperature in order to saturate the binding sites.
After washing the plates, serially diluted antiserum was added and
plates were incubated for 1 hour at room temperature followed by a
second wash with 0.5% Tween 20 in PBS. Peroxidase conjugated goat
anti-rabbit IgG, diluted 1:1000 in 1% BSA, was added and the plates
were again incubated at room temperature for 1 hour. After a final
washing, a color-forming substrate solution (O-phenylenediamine, 0.5 g
per 100 ml in 0.1 M citrate buffer pH 4.5 and 1.8% hydrogen peroxide)
was added, and the plates were incubated for 30 minutes at room
temperature. The absorbance at 492 nm wras measured with a Titertek
Multiscan reader. An absorbance of 0.05 or more over background was
considered positive. Background readings were obtained from the wells
in which all reagents except anti-5. gingivalis antiserum was added.
Normal rabbit serum was also tested against B. gingivalis antigen.
To test the effectiveness of adsorption, the titers of treated
sera were assayed as described above except that E. coli JM 109 (pUC9)
whole cells were used as the antigen.

21
Filter—Binding Enzyme immunoassay
Ampicillin-resistant transformants which formed white colonies in
the presence of X-Gal were spotted onto LB agar plates with arnpicillin,
grown overnight, and blotted onto nitrocellulose filter disks. B.
gingivalis and E. coli JM 109 (pUC 9) were also spotted onto each
filter as a positive and negative control, respectively. Duplicate
prints of the colonies on nitrocellulose filters were made and colonies
on one of each duplicate print were lysed by a 15-min. exposure to
chloroform vapor. Filters were then air dried for 30 minutes and
soaked for 2 hours in PBS containing 3% bovine serum albumin. After the
filters were urashed, adsorbed rabbit anti-5. gingivalis antiserum w^as
added and the filters were incubated in a solution of peroxidase
conjugated goat anti-rabbit immunoglobulin for 1 hour. After washing,
the filters were developed in a color-forming substrate solution
consisting of 0.06% 4-chloro-l- naphthol and 3% hydrogen peroxide in a
1:4 solution of methanol-TBS (50 mM Tris hydrochloride, 200 mM NaCl, pH
7.4). Clones which developed a blue color w-ere picked and rescreened
by the same procedure.
Restriction Analysis of Recombinant Plasmids
Plasmids were isolated from all the clones that were positive in
the filter-binding enzyme immunoassay. Restriction endonuclease
digestions were performed under conditions described by the
manufacturer to produce complete digestion. Agarose gel
electrophoresis was performed as described by Maniatis et al. (1982).
The size of DNA bands was estimated by comparing the distance of
migration to a logrithmic plot of the migration of standard restricted

22
lambda DNA run on the same gel.
Southern Plot Analysis
Recombinant plasmid and pUC 9 vector DNAs were digested to
completion with the appropriate restriction enzymes and run on a 1.2%
agarose gel. B. gingivalis DNA partially digested with Sau 3A, and Hind
III digested Eikenella corrodens clone 18 DNA (unpublished) were
also loaded in the gel. The DNA was transferred to Biodyne nylon
membrane by Southern transfer (Southern, 1975). B. gingivalis DNA
partially digested with Hind III was nick translated with ( a -32P
dCTP) (400 Ci/mmol, Amersham Corp., Arlington Heights, Ill.) as
described by Maniatis et al. (1982). The membrane-bound DNA w'as
O
hybridized to the nick-translated probe at 42 C in 50% formamide for 16
hours by the method recommended by the manufacturer (Pall Ultrafine
Filtration Corp., Glen Cove, N.Y.) which adapted from Wahl et al.
(1979). The membrane w'as washed at room temperature in wash buffer (2
O
x SSC and 0.1% SDS) four times each for 5 minutes and twdce at 50 C
each for 15 minutes in O.lx SSC, 0.1% SDS. An autoradiogram vras
obtained wúth Kodak XAR-5 film (Eastman Kodak Co., Rochester, N.Y.)
and Cronex Quanta II intensifying screen (Du Pont Co., Wilmington,
Del.).
Assay of the Titer of Ariti-ff. gingivalis Antiserum to E. coli
Transformants Which Express B. gingivalis Antigens
Cultures of each representative clone were prepared by 100 fold
O
dilution of overnight cultures and grown for 2 hours at 37 C.
Isopropyl- 6 -D-thiogalactopyrarioside (IPTG) was added to specific

23
cultures at a final concentration of 1 rnM and the ceils were pelleted
by centrifugation 4 hours later. The cells were washed, resuspended in
1/10 volume of PBS, and the optical density of each suspension was
determined at 550 rim. Cell lysate antigen was prepared by breaking the
cells with a sonicator. The protein concentration of each lysate was
determined by the Bio-Rad protein assay (Bio-Rad Laboratories,
Richmond, Calif.). Determination of the titer of anti-5, gingivalis
381 against these antigens was performed with the ELISA as described
above (10B cells or 1 pg protein per well). Normal rabbit serum
exhaustively adsorbed with E. coli JM109 (pUC9) was also tested in the
same manner.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and Western Blot Analysis of the Expressed Antigens
Each of the representative antigen-producing clones was grown to
mid-log phase in 3.0 ml of LB broth with 50 micrograms of ampicillin
per ml. The cells were pelleted, washed with PBS, resuspended in 0.3
ml of sample buffer (62.5 mM Tris-hydrochloride, 5% 2-mercaptoethanol,
2% SDS, 10% glycerol, 0.002% bromophenol blue, pH 6.8), and boiled for
3 minutes. The B. gingivalis cell lysate was mixed with an equal
volume of sample buffer and treated in the same manner. SDS-PAGE was
performed in a vertical slab gel electrophoresis tank (Hoefer
Scientific Instruments, San Francisco, CA.) as described by Laemmli
(1970). Samples of 0.03 ml from each clone as well as 40 micrograms of
Bacteroides cell lysate were run at a constant current of 20 mA per gel
through the 4% polyacrylamide stacking gel (pH 6.8) and 30 mA per gel
through the 12.5% or 5% separating gel (pH 8.3). The gels were

24
processed either by staining with Coomassie brilliant blue R2-"1
(Fairbanks et al., 1971), or used for Western blot analysis.
Western blotting was done as described by Burnette (1981) as follows.
Separated antigens on the gel were transferred to nitrocellulose paper
(0.45 m) (Schleicher & Schuell Co., Inc., Keene, NH) by
electroblottirig using the Hoefer apparatus at 60 V overnight with a
buffer containing 20 mM Tris base, 150 mM glycine, and 20% methanol (pH
8.3). The blot was visualized as follows. Nitrocellulose sheets were
preincubated in a blocking solution of PBS with 2% BSA and 0.1%
Tween-20 for 2 hours or overnight. Adsorbed antisera used as probes
were usually diluted 1:100 in blocking solution and reacted with the
nitrocellulose transfer for 1.5 hours. After w'ashing with distilled
water, the membranes were incubated with affinity purified goat
anti-rabbit IgG for 1.5 hours. After washing again with distilled
water, the membranes w^ere developed in the color-forming substrate
solution as described in the filter-binding enzyme immunoassay. The
molecular weight of each individual band wras estimated by comparison to
the molecular weight standard proteins run on the same gel.
Results
Titer of Antisera
Rabbit anti-B. gingivalis antiserum had an antibody titer of 1:
64,000 to B. gingivalis and 1:160 to E. coli (pUC 9), whereas
normal rabbit serum had an antibody titer of 1:10 to B. gingivalis and
1:80 to E. coli (pUC 9). Adsorption of anti-B. gingivalis antiserum
with E. coli (pUC 9) resulted in a slight reduction of antibody titer

25
to B. gingivalis and reduced the anti-¿s', coli titer to zero or 1:10.
Identification of E. coli Tra:¡sformants Which Expressed B. gingivalis
Antigens
Approximately 4,500 tranformants generated from the Sau 3A restricted
chromosomal DNA were tested for the expression of B. gingivalis
antigens by the filter-binding enzyme immunoassay using E. coli-
adsorbed rabbit anti-B. gingivalis serum. Only 1 clone (clone 3)
was positive when either lysed or unlysed cells were tested. A total
of 1,700 colonies of transformants resulting from Hind III restricted
chromosomal DNA were also tested for the expression of B. gingivalis
antigens. Seven clones gave positive signals. Of these 7 clones, one
was positive only when lysed (clone 8) and the rest were positive both
when lysed and unlysed (Table 1).
Agarose Gel Electrophoresis of Recombinant Plasmids
To further confirm the positive results of the filter-binding
enzyme immunoassay, plasmid DNA was isolated from each positive clone.
Electrophoresis of these unrestricted plasmids showed that each clone
contained only one recombinant plasmid (Figure 2, lanes 1 through 8).
Clone 3, which was constructed by ligation of Sau 3A partially
digested B. gingivalis DNA with Bam HI cut pUC 9, could not be digested
with Bam HI (Figure 3, lane 10). Restriction of pUC 9 with enzyme Sma
I and Sal I deletes a 9 bp fragment containing the Bam HI site from pUC
9 (Figure 2, lane 18 and Figure 3, lane 4, see Figure 1 for map of pUC
9). Therefore, clone 3 DNA w-as restricted with Sma I and Sal I.
Restriction analysis revealed a fragment of linear 9 bp-deleted pUC 9

26
Table 1. Characterization of E. coli transformants
which express B. gingivalis antigens
Colonies reacted
Size of B.
Clone No.
with antiserum
unlysed lysed
gingivalis
DNA cloned (Kb)
1 and 2
+ a
+
3.2
3
+
+
1.1
4
+
+
3.3
5 and 6
+
+
5.5
7
+
+
4.8
8
— b
+
3.5
a = Positive reaction
b = Negative, not reactive

Figure 2. Agarose gel electrophoresis of recombinant plasmids.
Lanes: 1-8, undigested recombinant plasmids from clones 1 - 8; 9, pUC 9
digested with Hind III; 10 - 13, recombinant plasmids from clones 5, 6, 7, and 8
digested with Hind III; 14, pUC 9 digested with Pvu II; 15 - 17, recombinant
plasmids of clones 1, 2, and 4 digested with Pvu II; 18, pUC 9 digested with Sma
I and Sal I; 19, recombinant plasmid of clone 3 digested with Sma I and Sal I.

28

29
and 2 fragments of insert (Figure 2, lane 19 and Figure 3, lane 5).
Restriction analysis with different enzymes (Figure 3) showed that the
size of insert of clone 3 was approximately 1.1 kb.
Clones 1, 2, 4, 5, 7, and 8 were generated from Hind Ill-
restricted chromosomal DNA. After digestion with Hind III, only clones
5, 6, 7, and 8 revealed fragments of the linear pUC 9 vector and
fragments of B. gingivaJis DNA inserts (Figure 2, lanes 10 through 13).
Plasmid DNAs of these clones were restricted with various enzymes and
analyzed by gel electrophoresis (Figure 4). The estimated siz- of
inserts of clones 5, 6, 7, and 8 are 5.5, 5.5, 4.8, and 3.5 kb,
respectively (Table 1). Thus clones 5 and 6 were found to contain
plasmids of the same size and identical restriction fragments.
Although clones 1, 2, and 4 were generated from Hind III
restricted DNA, they did not result in fragments of linear pUC 9 after
Hind III digestion (Figure 5, lanes 6, 11, and 16). These cloned DNAs
were then restricted with Pvu II, which generates a 307 bp fragment
containing the polylinker-cloning sites from pUC 9 (Figure 1 and Figure
2, lane 14 and Figure 5, lane 4). Clones 1, 2, and 4 revealed
fragments of linear 307 bp-deleted pUC 9 and inserts associated w-ith
the deleted fragment (Figure 2, lanes 15, 16, and 17). These cloned
DNAs were digested wdth various restriction enzymes and analyzed by
agarose gel electrophoresis (Figure 4). The size of inserts of clones
1, 2, and 4 were estimated to be 3.2, 3.2, and 3.3 kb, respectively
(Table 1). Clones 1 and 2 also contained plasmids of the same size and
identical restriction fragments.

Figure 3. Agarose gel electrophoresis of different restriction digests of the
recombinant plasmid from clone 3.
Lanes: 1, DNA marker-Hind 111/Eco RI digest of lambda DNA; 2, undigested pUC 9;
3, pUC 9 digested with Hind III; 4, pUC 9 digested with Sma I and Sal I; 5, 6, 7,
8, 9, and 10, recombinant plasmid from clone 3 digested with Sma I and Sal I, Sma I
alone, Sal I alone, Hind III, Eco RI, and Bam HI, respectively; 11, undigested
recombinant plasmid from clone 3; 12, DNA marker-Hind III digest of lambda DNA.


Figure 4. Agarose gel electrophoresis of different restriction digests of
recombinant plasmids from clones 5, 6, 7, and 8.
Lanes: 1, DNA marker-Hind III/Eco RI digest of lambda DNA; 2, pUC 9 digested
with Hind III; 3, 4, and 5 recombinant plasmid from clone 5 digested with Hind III,
Eco RI and Bam HI respectively, 6, 7, and 8, recombinant plasmid from clone 6 digested
with Hind III, Eco RI and Bam HI, respectively; 9, 10, and 11, recombinant plasmid
from clone 7 digested with Hind III, Eco RI and Bam HI, respectively; 12, 13, and 14,
recombinant plasmid from clone 8 digested with Hind III, Eco RI and Bam HI,
respectively; 15 - 18, undigested recombinant plasmids from clones 5-8; 19, DNA
marker-llind III digest of lambda DNA.


Figure 5. Agarose gel electrophoresis of different restriction digests of
recombinant plasmids from clones 1, 2, and 4.
Lanes: 1, DNA marker-Hind III/Eco RI digest of lambda DNA; 2, pUC 9 undigested;
3, pUC 9 digested with Hind III; 4, pUC 9 digested with Pvu II; 5, 6, 7, and 8,
recombinant plasmid from clone 1 digested with Pvu II, Hind III, Eco RI and Bam HI,
respectively; 9, undigested recombinant plasmid from clone 1; 10, 11, 12, and 13,
recombinant plasmid from clone 2 digested with Pvu II, Hind III, Eco RI, and Bam HI,
respectively; 14, undigested recombinant plasmid from clone 2; 15, 16, 17, and 18,
recombinant plasmid from clone 4 digested with Pvu II, Hind III, Eco RI, and Bam HI,
respectively; 19, undigested recombinant plasmid of clone 4.


36
Hybrid ,nation of Recombinant Plasmids rith D. gingivalis DXA Probe
Southern t iot analysis was also performed to confirm that the DNA
inserts were derived from the P. gingivalis DNA. As can be seen in
Figure 6, the hybridization pattern of most of the insert fragments
showed dark bands of homology to the B. gingivalis chromosomal DNA
probe. The pUC 9 showed a faint band with homology to the probe.
Increasing the stringency of the wash (65° C for 1 hour) did not
significantly change the hybridization pattern. How'ever, a shorter
exposure of the autoradiograph eliminated the background of pUC 9 but
the tu:o smallest insert bands from clone 4 also disappeared. The
control DNA from Eikenella corrodens did not hybridize with the P.
gingivalis DNA probe (Figure 6, lane 12).
Titer of Anti-13. gingivalis Antiserum to E. coli Transformants
Anti-B. gingivalis antiserum was able to detect antigen expression
in all positive clones except clone 8 in an enzyme-linked immunosorbent
assay (ELISA) (Table 2). The antiserum reacted with both whole cell
and cell lysate antigens. Isopropyl- 8-D-thiogalactopyranoside (IPTG)
was not necesary to induce antigen expression. However, in the
presence of IPTG, clones 2 and 3 showed higher antigen expression,
especially when the cell lysate preparations were tested.
Determination of the Expressed Antigens in E. coli JM 109
Five stable representative clones were analyzed for antigen
expression by SDS-PAGE and Western blot analysis. As can be seen in
Figure 7, only clones 2 and 3 produced antigens detectable by E. coli
adsorbed anti-5, gingivalis antiserum in the Western blot. Antigens

32
Figure 6. Hybridization of recombinant plasmids with P labeled B. gingivalis
DNA probe.
A. Agarose gel electrophoresis of DNA before Southern transfer.
Lanes: 1, Sau 3A partially digested B. gingivalis DNA; 2, pUC 9 digested with Pvu II;
3, recombinant plasmid from clone 1 digested with Pvu II; 4, recombinant plasmid from
clone 2 digested with Pvu II; 5, recombinant plasmid from clone 4 digested with Pvu II;
6, pUC 9 digested with Hind III; 7, recombinant plasmid from clone 5 digested with Hind
III; 8, recombinant plasmid from clone 6 digested with Hind III; 9, recombinant plasmid
from clone 7 digested with Hind III; 10, recombinant plasmid from clone 3 digested with
Sma I and Sal I; 11 and 12, recombinant plasmid from Eikenella corvodens clone 18
digested with Hind III.
B. Autoradiograph of Southern blot hybridization of the agarose gel in panel A.


39
Table 2. Titer of anti-5, gingivalis antiserum against E. coli
transformants which express B.gingivalis antigens
Antibody titers®
against test antigensb
Organism
Whole cell
Cell lysate
IPTG- IPTG+
IPTG- IPTG+
Clone 1
320
NTC
320 - 640
NT
2
320
640
320 - 640
1,280 - 2,560
3
20
160
40 - 160
1,280
4
20 - 100
20 - 40
20 - 40
20 - 40
5
40 - 80
40 - 80
40 - 80
40 - 80
6
40
NT
40
NT
7
40
40
40
40
8
0
0
0
NT
E. coli
JM 109 (pUC
9) 0-10
0-10
0-10
0-10
B. gingivalis
40,950 - 64,000
NT
NT
NT
Control NRSd
a Number designates the reciprocal dilution of the sera which gave
OD492 reading of 0.05 or more over the background. Antiserum was
exhaustively adsorbed with E. coli JM109 (pUC 9).
b Antigens were prepared from cultures grown without IPTG (IPTG~)
or in the presence of IPTG (IPTG+>-
c Not tested.
d Normal rabbit serum exhaustively adsorbed with E. coli JM 109
(pUC 9) did not react to test antigens.

Figure 7. SDS-PAGE on 12.5% acrylamide and Western blot analysis of expressed
B. gingivalis antigens. Molecular weight standards (Pharmacia Fine Chemicals,
Piscataway, N.Y.) are phosphorylase b (94 K, 94,000 molecular weight), albumin
(67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor
(20,100) and a-lactalbumin (14,400).
Lanes: 1, B. gingivalis cell lysate (40 ng); 2 to 7, whole cell samples of clones
2, 3, 4, 5, 7, and E. ooli JM 109 (pUC 9) as described in the methods.
A. The gel after Coomassie blue stain. B. The blot was probed with E. ooli-
adsorbed anti-B. gingivalis antiserum. C. The blot was probed with normal
rabbit serum.


ñ o
expressed in clones 4, 5, and 7 were not detected by Western blot
analysis. Normal rabbit serum reacted to some common antigens among
these clones and E. coli JM 109 (pUC 9). The anti-B. gingivalis
antiserum did, however, react with a protein band of approximately
140,000 (140 K), as well as a smear of lower molecular weight from
clone 2. Multiple bands of 30 to 50 K from clone 3 were also detected.
These particular polypeptides were not detectable in E. coJi JM 109
(pUC 9) preparations (Figure 7, lane 7). A whole cell preparation from
clone 2 was also separated in a 5% SDS polyacrylamide gel and the
expressed protein w'as estimated to have a molecular u'eight of 125 K
(Figure 8).
Discussion
Genomic libraries of B. gingivalis DNA were constructed in the
plasmid expression vector pUC 9, which contains the pBR 322 origin of
replication, the pBR 322 ampicillin resistance gene, and a portion of
the lac Z gene of E. coli which codes for the a -peptide of
8-galactosidase (Figure 1). The amino terminus of the lac Z gene
contains a polylinker region wrhich has multiple unique cloning sites.
Transformation of E. coli JM 109, which is defective in 8-
galactosidase, with pUC 9 complements the bacterial 8 -galactosidase
activity, resulting in the ability of the bacterial cell to metabolize
the lactose analog X-Gal to a blue color. Cloned DNA inserted in the
polylinker region will interrupt the lac Z gene of the plasmid.
Therefore E. coll transformants resulting from recombinant plasmids will
be unable to metabolize X-Gal and appear as u'hite colonies on X-Gal
containing plates. The advantages to this plasmid are 1) DNA inserted

Figure 8. SDS-PAGE (on 5% acrylamide) of expressed B. gingivalis
antigen in clone 2.
Lanes: A) Molecular weight standards (Sigma Chemical Co., St.
Louis, Mo.) are myosin (205 K, 205,000), g-galactosidase (116,000),
phosphorylase B (97,400); B) Whole cell sample of clone 2.

44
205 K
r-Á

45
into any of the cloning sites, which are downstream from a strong
promoter, should be expressed whether or not a B. gingivalls promoter
is cloned with a structural gene, 2) transformants containing a
recombinant plasmid are easily detected upon initial selection, and 3)
the multiple cloning sites make it a versatile cloning vector which is
especially useful for subcloning.
Five different E. coli clones stably exhibited B. gingivalis
antigen expression. These antigens were detected in intact cells both
by filter-binding enzyme immunoassay (Table 1) and ELISA (Table 2).
Although it has not yet been confirmed by immunoeleetronmicroscopy, it
is likely that these Bacteroides antigens are located on the E. coli
cell surface, and therefore must contain a leader peptide in order to
be translocated to the E. coli surface (Oliver, 1985). This result
suggests that B. gingivalis surface antigens can be processed as well
as expressed in E. coli.
Clones 1 and 2 have undergone some kind of DNA rearrangement,
i.e., the recombinant plasmids, 'when cut by Hind III, did not result
pUC 9 and insert band but showed one large band and one small band
(Figure 2, lanes 10 and 11). This apparent rearrangement may result
from a deletion at one Hind III end of the insert and another Hind III
end may still be intact.
Clone 2 w-as found to encode a polypeptide with an average
molecular weight of 125 K, seen in polyacrylamide gels and detected by
Western blot analysis (Figures 7 and 8). The smear at the lower
molecular weight seen in the blot may be the degraded product of this
expressed antigen, since E. coli has a functional Ion gene which
encodes for the enzyme involved in degradation of internal abnormal

46
proteins (Charette et al., 1981; Chung and Goldberg, 1981; Waxman and
Goldberg, 1982).
The function of the lac promoter in pUC 9 does not depend on IPTG;
it is, however, enhanced by IPTG, since E. coli JM 109 (pUC 9) grows as
blue colonies on medium containing X-Gal in the absence of IPTG.
Expression of the B. gingivalis antigen in clone 2 occurs either in the
presence or absence of IPTG but is enhanced by IPTG stimulation. This
result suggests that the direction of transcription of this DNA insert
is the same as that of g-galactosidase and is likely to be under the
control of the lac promoter. Assuming an average molecular weight of
100-125 for an amino acid, the insert of clone 2, estimated to be
3,200 bp, could encode for a 125 K polypeptide. The expressed
polypeptide may be fused to the major portion of the ot-peptide of
8-galactosidase w’hich would add approximately 100 amino acids to the
expressed polypeptide. The expression of the clone 3 antigen was also
found to be stimulated by IPTG in the same manner as clone 2. The
size of the clone 3 insert (1,100 bp) is large enough to encode for the
expressed antigen (30 to 50 K) observed by Western blotting.
The synthesis of Bacteroides antigens in clones 4, 5, and 7 was
not found to depend on the presence of IPTG or to be enhanced by IPTG
(Table 2). This suggests that a functional Bacteroides promoter is
included with the structural gene of each clone. However, antigen
expression of these clones cannot be detected by Western blot
analysis. This might be due to 1) the antigens not being transferred
to the nitrocellulose sheets, 2) the transferred antigens containing
altered conformations which are not recognized by the antiserum, or 3)
the antigen expression being too low to be detected.

47
These results have demonstrated that the B. gingivalis genome
can be cloned and expressed in E. coli. The cloned antigens are
presently being identified and further characterized for functional
properties. The cloning of B. gingivalis genes is an approach that
provides new tools for investigations into the pathogeneeity of B.
gingivalis.

CHAPTER THREE
CHARACTERIZATION OF BACTEROIDES GINGIVALIS
ANTIGENS SYNTHESIZED IN ESCHERICHIA COLI
Introduction
Bacteroides gingivalis possesses several potential virulence
factors which may 1) promote its colonization in the host, 2) resist
host defenses, and 3) cause destruction of periodontal tissues (Slots
and Genco, 1984). Colonization, the initial event in the establishment
of disease, requires the adherence of bacteria to host tissues (Gibbons
and Van Houte, 1975), therefore bacterial surface components which
mediate bacterial adherence are considered to be important virulence
factors. In the oral cavity, bacteria can attach to host tissues as
well as to bacteria in pre-formed plaque (Slots and Gibbons, 1978).
The nature of the binding sites on teeth and oral tissues to u'hich
periodontopathic bacteria, including B. gingivalis, attach has not been
well established. In vitro, B. gingivalis can attach to and
agglutinate erythrocytes (Okuda and Takazoe, 1974; Slots and Gibbons,
1978; Slots and Genco, 1979; Okuda et al., 1981), can adhere in high
numbers to human buccal epithelial cells (Slots and Gibbons, 1978;
Okuda et al., 1981), to crevicular epithelial cells derived from
periodontal pockets (Slots and Gibbons, 1978), and to surfaces of Gram
positive bacteria present in plaque, (Slots and Gibbons, 1978; Schwarz
et al., 1987). In addition it will adhere to untreated and saliva-
treated hydroxyapatite (SHA), but in comparatively low numbers (Slots
and Gibbons, 1978). B. gingivalis has also been reported to bind to
48

49
HR9 matrix, a material similar to the basement membrane barrier
underlying connective tissue (Leong et al., 1985). Recently, it has
been reported that B. gingivalis can bind to fibrinogen and possibly
colonize host tissue by attaching to fibrinogen-coated surfaces (Lantz
et al., 1986).
Since the components involved in B. gingivalis adherence in vivo
are, at present, ill defined, the expression of any structure detected
by in vitro methods thus needs to be examined. Therefore, the
antigen-expressing clones described in Chapter Two were tested for the
expression of adhesins for saliva-treated hydroxyapatite (SHA adhesin)
and erythrocytes (hemagglutinin). This chapter describes the assay for
the SHA adhesin by testing for removal of SHA adherence inhibition by
anti-B. gingivalis antiserum and the assay for hemagglutinin by a
direct hemagglutination test. The clones which were able to
agglutinate erythrocytes 'were analyzed by restriction analysis of their
B. gingivalis DNA inserts and DNA homologies were tested by Southern
blot hybridization. Antibodies against these clones were made in
rabbits and used as probes to identify the native antigens of B.
gingivalis by Western blot hybridization. B. gingivalis DNA inserts
from clones 2 and 7 were used as probes in the hydridization of several
restricted B. gingivalis chromosomal DNAs to determine whether these
inserts are adjacent to each other in the chromosomal DNA.

50
Materials and Methods
Bacterial Strains and Growth Conditions
Bacteroides gingivalis 381 was cultured in Todd-Hewitt broth as
described in Chapter Two. E. coli transformants were cultured in LB
medium containing 50 micrograms of ampicillin per ml by preparing 100
fold dilutions of overnight cultures followed by incubation for 2 hours
O
at 37 C. IPTG was added to the cultures, when used at a final
concentration of 1 mM, and the cultures were incubated for an
additional 4 hours.
Assay for Removal of SHA Adherence Inhibition by Anti-13. gingivaJis
Antiserum
Aliquots of anti-B. gingivalis antiserum were adsorbed with each
antigen-expressing clone as well as E. coli JM 109 (pUC 9) as described
in Chapter Two. The titer of each adsorbed antiserum was tested
against ee h clone and B. gingivalis whole cell antigen by ELISA as
described above.
Whole paraffin-stimulated human saliva was collected and heated at
O
56 C for 30 minutes to inactivate degradative enzymes. Extraneous
debris and cells were removed by centrifugation at 12,000 rpm for 10
minutes and sodium azide wras added to a final concentration of 0.04%.
Hydroxyapatite beads (HA) (BDH Biochemical, Ltd., Poole, England)
urere treated as previously described (Clark et al., 1978). Briefly, 10
mg of beads were washed and hydrated in distilled water in 250
microliter plastic microfuge tubes followed by equilibration overnight
with adsorption buffer (0.05 M KC1, 1 mM K2HPO4, pH 7.3, 1 mM CaCl2

51
and 0.1 mM MgCk). The beads were incubated with 200 microliters of
saliva for 24 hours at 4°C and then washed with sterile adsorption
buffer to remove nonadsorbing material. Control tubes without HA were
treated identically.
B. gingivalis 381 cells were labeled by growth to late log phase
in medium supplemented with (3H) thymidine (10 mCi/ml). The cells
were pelleted, washed twice in adsorption buffer, and dispersed with
three 10-second pulses (medium power) with a microultrasonic cell
disrupter.
The dispersed cells were mixed with each antiserum (1:100
dilution) and normal rabbit serum to a final concentration of 4 x 106
cell/ml. The cell-antiserum suspensions (200 microliters) wrere then
added to the SHA beads in microfuge tubes and the tubes were rotated in
an anaerobic chamber for 1 hour. Labeled cells alone (no antisera)
were treated in the same manner to determine the number of cells
adhering to the SHA surface. A control tube containing cells but no
SHA wras tested to quantitate the amount of cells bound to the tubes
rather than to the SHA. One hundred microliters of adsorption buffer
containing unadhered cells wras removed and placed in minivials
containing 3 ml of aqueous scintillation cocktail (Amersham/Searle,
Arlington Heights, IL), and counted with a Scintillation Counter (Model
455 Parkard Tricarb). Determination of the number of cells adhering to
the SHA was done by subtracting the number of cells (no. of counts) in
solution from the total number of cells (no. of counts) which did not
adhere to the tube.

52
Direct Hemagglutination Assay
The hemagglutination assays were carried out in V-bottom
microtiter plates (Dynatech Laboratories, Inc., Alexandria, Virginia).
Erythrocytes (sheep or human group 0) were washed 3 times with PBS
(0.02 M phosphate buffered saline), pH 7.2, and resuspended to a final
concentration of 0.2% (v/v). Cells of B. gingivalis and antigen¬
expressing clones were washed twice in PBS and resuspended to an
optical density of 0.5 and 2.0, respectively, at 660 rim. The cell
suspensions were diluted in a twofold series with PBS and 0.05 ml of
the suspensions were added to the wells. E. coli JM 109 (pUC 9) which
was prepared in the same manner as the antigen-expressing clones, was
included as a control. An equal volume (0.05 ml) of washed
erythrocytes was added and mixed with the bacterial cells. The plates
were stored for 16 hours at 4 °C and then examined for evidence of
hemagglutination as follows. Agglutinated erythrocytes will settle as
clumps which will be dispersed throughout the bottom of the wells,
resulting in a pinkish-red coating of each well. In the absence of
hemagglutination, the erythrocytes will settle on the bottom of the
well as a central, smooth, bright red round disk. The titer was
expressed as the reciprocal of the highest dilution showing positive
agglutination.
Hemagglutination Inhibition Assay
The hemagglutination inhibition assay was also carried out in V-
bottom microtiter plates. B. gingivalis cell suspensions in PBS u'ere
adjusted to the optical density of 0.5 at 660 nm. Each antiserum
examined for hemagglutination inhibition activity was diluted two-fold

53
in a series of wells. Fifty yl of the bacterial suspension with twice
the minimum number of cells which produced hemagglutination was then
added to each well. After incubation with gentle shaking at room
temperature for 1 hour, 0.05 ml of the w'ashed erythrocytes were added
to each well and mixed. The plates are left for 16 hours at 4 C and
read for hemagglutination as described above for the hemagglutination
assay. The titer was expressed as the reciprocal of the highest
dilution showing hemagglutination inhibition.
Preparation of Antisera to Hemagglutinable E. coli
E. coli transformants which were able to agglutinate erythrocytes
were grown in LB broth containing ampicillin as described above. Tw'o
rabbits w'ere injected wdth each clone as described in Chapter Two.
Sera were exhaustively adsorbed with E. coli JM 109 (pUC 9) and tested
for anti-i?. ginglvalis activity by ELISA.
Adsorption of Anti-Clone 2 Antiserum
Anti-clone 2 antiserum diluted 1:10 was separately adsorbed with B.
gingivalis, E. coli JM 109 (pUC 9), and clones 2, 5, and 7. Washed
stationary phase cells of each bacterial culture were prepared as
described in Chapter Twro. For each adsorption, 107, 108, 109, and 1010
bacterial cells were mixed with 200 ul of serum and the suspensions
were stored at 4°C overnight. The sera were recovered by
centrifugation at 12,000 g for 10 minutes. Each adsorbed antiserum was
assayed by ELISA to determine the titer to B. gingivalis.

54
DNA Procedures
Restriction endonuclease digestions of the recombinant plasmids
from clones 2, 5, and 7 were performed according to manufacturer's
directions. The size of DNA inserts were estimated and Southern blot
analysis was performed as described in Chapter Two. Clone 5 DNA was
digested with Hind III and two fragments of B. gingivalis inserts were
isolated from agarose gels by the method of Zhu et al. (1985) employing
centrifugal filtration of DNA fragments through a Millipore membrane
inside a conical tip. The DNA preparations were extracted with phenol-
chloroform, precipitated with ethanol and resuspended in TE, pH 8.0.
Each DNA fragment was ligated to Hind III digested pUC 9 and the
resulting recombinant plasmids were transformed into competent E. coli
JM 109 cells as described in Chapter Two. Recombinant plasmids from
these transformants were isolated by rapid plasmid DNA isolation
(Silhavy et al., 1984), digested with appropriate restriction
endonucleases, and analyzed by agarose gel electrophoresis.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
and Western Blot Analysis
B. gingivalis cell lysate and cells of E. coli transformant wrere
prepared and analyzed by SDS-PAGE and Western blot techniques as
described in Chapter Two. Antisera to clones 2, 5, and 7 exhaustively
adsorbed with E. coli JM 109 (pUC 9) were used as probes in the Western
blot. Control antisera included anti-clone 2 antiserum also adsorbed
with B. gingivalis at the ratio of 1010 cells per 100 ul of antiserum,
and antiserum to E. coli JM 109 harboring pUC 9 with Eikenella
corrodens DNA insert.

55
Results
Assay for SHA Adhesin
It is possible that if the B. gingivalis SHA adhesin is expressed
in E. coli, it may be expressed in a functionally inactive form, due to
spacial interference by other E. coli surface structures, or it may not
be processed adequately by the E. coli protein translocation machinery
and thus may not be properly expressed on the E. coli surface. However,
there is a strong possibility that the expressed antigen would still be
antigenically intact. Thus, anti-B. gingivalis 381 antiserum which
inhibits the adherence of B. gingivalis 381 to SHA was adsorbed with
each antigen-expressing clone until the titer of this antiserum to each
clone was reduced to zero. Each adsorbed antiserum was tested for
inhibition of B. gingivalis adherence to SHA. If a clone expresses an
antigenically active adhesin, the adsorbed antiserum should be unable
to inhibit B. gingivalis 381 adherence to SHA or may partially inhibit
the adherence.
The results in Table 3 summarize the SHA inhibition data and
indicate that the antiserum adsorbed with each antigen-expressing clone
still inhibited the adherence of B. gingivalis. There is no apparent
significant decrease in the percent inhibition by each adsorbed
antiserum.
Assay for Hemagglutinin
The rationale to identify the clones which express hemagglutinin
were analogous to those described for the SHA adhesin. The anti-B.
gingivalis antiserum adsorbed with each antigen-expressing clone and E.

56
Table 3. Inhibition of adherence to SHA by adsorbed
anti-5, gingivalis antisera
Inhibitor and dilution
% adherence8
% inhibition11
None
83.35
Normal rabbit serum
1:100
80.08
0.05
Antiserum unadsorbed
Antiserum adsorbed with:
1:100
22.70
72.15
E. coli JM 109 (pUC 9)
1:100
21.57
73.07
Clone 2
1:100
10.73
86.59
Clone 3
1:100
22.60
71.78
Clone 4
1:100
16.24
79.71
Clone 5
1:100
27.37
65.82
Clone 7
1:100
19.90
75.15
a Percent adherence was calculated from the following formula:
% adherence = [ (CPM from tube without SHA - CPM from tube with
SHA)/(CPM from tube without SHA)] x 100.
b Percent inhibition was calculated from the following formula:
% inhibition = Cl - (% adherence in the presence of antibody / %
adherence in the absence of antibody)] x 100.

57
coli JM 109 (pUC 9), as described for the SHA assay, were tested for
removal of hemagglutination inhibition activity of anti-B. gingivalis
antiserum. Since it is necessary to determine the minimum number of B.
gingivalis cells which produces hemagglutination before performing the
hemagglutination inhibition assay, a direct hemagglutination assay of
antigen-expressing clones together with B. gingivalis was first
performed.
The direct hemagglutination assay of these clones demonstrated
that clones 2, 5, and 7 did agglutinate sheep erythrocytes, whereas E.
coli JM 109 (pUC 9) did not (Figure 9). The hemagglutination titer of
clone 2 was 2 and that of clones 5 and 7 agglutinated erythrocytes at
the undiluted suspension. In addition, clone 5 was found to auto-
agglutinate when resuspended in PBS, pH 7.2.
Restriction Maps
Since three of the antigen-expressing clones were found to
agglutinate erythrocytes, the possibility arose that they may have
common DNA inserts which encode the same function. Clone 2 resulted
from some kind of DNA rearrangement, i.e., clone 2 DNA when cut by Hind
III, did not result in pUC 9 and insert bands but showed one large band
and one small band as described in Chapter Two (Figure 2, lane 11).
The rearrangement may have resulted from a deletion or other
rearrangement at one Hind III end of the insert and another Hind III
end may still be intact. In order to obtain information as to the
nature of the rearrangement of clone 2 and the relationship of the
three hemagglutinating clones to one another, restriction maps of these
three clones were generated.

Figure 9. Hemagglutination of sheep erythrocytes. Bacterial suspensions were
diluted in 2 fold serial dilutions and mixed with an equal volume of 0.2% (V/V)
erythrocytes as described in the methods. Row 1 is the undiluted bacterial suspensions.
(A) B. gingivalis 381, (B) E. ooli JM 109 (pUC 9), (C) clone 2, (D) Clone 3,
(E) Clone 4, (F) Clone 5, (G) Clone 7.

59

60
The recombinant plasmids of clones 2, 5, and 7 were restricted
with several restriction endonucleases and analyzed in 1.2% agarose
gels as shown in Figures 10, 11, arid 12. A restriction map of each
clone was generated as shown in Figures 13, 14, and 15. A schematic
diagram of restriction enzyme recognition sites of these three clones
is detailed in Figure 16. This data indicates that the clone 2 insert
appears to be different from that of clones 5 and 7, whereas clones 5
and 7 have one insert fragment in common. The restriction map of clone
2 revealed that the Hind III site of the DNA insert at the amino
terminal end of the 3-galactosidase gene was still intact but a
deletion occurred at the other end of the insert and included most of
the linker. The linker region with recognition sites of Pst I, Sal I,
Bam HI and Sma I was deleted but the Eco RI site was still intact as
wrell as other sites upstream such as Pvu II and Nar I.
Southern Blot Analysis
To further confirm the results of the restriction maps,
32P-labeled clone 7 recombinant DNA was used as a probe for
hybridization of restricted recombinant plasmids by Southern blot
analysis. Clone 2 DNA restricted with Hind III, Eco RI, and Sma I
resulted in DNA fragments of pUC 9 and 4 pieces of insert of
approximately 1,400, 1,300, 420, and 150 bp (Figure 17, panel A, lane
2). Clone 5 DNA restricted with Hind III resulted in fragments of pUC
9 and 2 pieces of insert of approximately 4,800 and 760 bp (Figure 17,
panel A, lane 3). Fragment bands of pUC 9 and inserts of approximately
2,800, 2,000, and 760 bp were generated from digestion of clone 5 DNA
with Hind III and Bam HI (Figure 17, panel A, lane 5). Clone 7 DNA

Figure 10. Agarose gel electrophoresis of restriction digests of the recombinant
plasmid from clone 2.
Lanes: 1, DNA marker-Hind III digest of lambda DNA; 2, pUC 9 digested with Hind III;
3, pUC 9 digested with Pvu II; and recombinant plasmid from clone 2 digested with;
4, Hind III; 5, Pvu II; 6, Pvu II and Hind III; 7, Eco RI; 8, Eco RI and Hind III;
9, Sma I; 10, Sma I and Hind III; 11, Sma I and Eco RI; 12, Eco RI and Pvu II; 13,
Nar I; 14, Eco RI and Nar I; 15, Bam HI; 16, Hind III and Bam HI; 17, Sma I and
Bam HI; 18, Cía I; 19, Cía I and Hind III; 20, Eco RI and Cía I.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 11. Agarose gel electrophoresis of restriction digests of the recombinant
plasmid from clone 5.
Lanes: 1, DNA marker-Hind III digest of lambda DNA; 2, pUC 9 digested with Hind III
and recombinant plasmid from clone 5 digested with; 3, Hind III; 4, Bam HI; 5, Hind
III and Bam HI; 6, Sal I; 7, Hind III and Sal I; 8, Hind III and Eco RV; 9, Stu I;
10, Hind III and Stu I; 11, Stu I and Sal I; 12, Stu I and Eco RV; 13, Stu I and
Asp 718; 14, Hind III and Asp 718; 15, Bam HI and Asp 718; 16, Bam HI and Stu I;
17, Eco RI; 18, Hind III and Eco RI.

23.1
9.4
6.5
4.3
2.3
2.0
1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 18
en
0.5

Figure 12. Agarose gel electrophoresis of restriction digests of recombinant
plasmids from clones 5 and 7.
Lanes: 1, DMA marker-Hind III digest of lambda DNA; 2, pUC 9 digested with Hind III;
recombinant plasmids from clones 5 and 7 in adjacent lanes digested with; 3 and 4,
Hind III; 5 and 6, Hind III and Bam HI; 7 and 8, Hind III and Asp 718; 9 and 10,
Hind III and Stu I; 11 and 12, Sal I; 13 and 14, Hind III and Eco RI.

23.1
9.4
6.5
4.3
2.3
2.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
CT>
CTi

Figure 13. REstriction map of the recombinant plasmid from
clone 2. The heavy line represents B. gingivalis DNA insert.

Hind III
Bam HI
Hind HI
Bam HI
Cía I
Sma I
Nar I
Cía I
Sma I
Nar I
EcoRI
o
3,200 3,000 2,000 1,000

Figure 14. Restriction map of the recombinant plasmid from
clone 5. The heavy line represents B. gingivalis DMA insert.

Hind III
Eco RV
Sal I
Stu I
Hind III
Bam HI
Asp 718
Stu I
Eco Rl
â–  Hind III
— Pst I
— Sal I
■—Bam HI
— Sma I
•Eco Rl
o
o
o
I— o
cr
•O
o

Figure 15. Restriction map of the recombinant plasmid from
clone 7. The heavy line represents B. aingivalis DNA insert.

Hind III
Pst I
Sal I
Bam HI
Sma I
Eco Rl
cr
â– o
o
4.800 4,000 3,000 2,000 1.000

Figure 16. Schematic diagram of restriction enzyme recognition sites of recombinant
plasmids from clones 2, 5, and 7. The solid lines represent B. gingivalis DNA
inserts. The hatched boxes represent pUC 9 regions.

/ 3NO10 '//////AYA—i i—n YA'/Z/Z///.
'«0 ?
ia
0/
a,
'*D
«A
ia
o/
— Cía I
Sma I
iq-N,r-k]—«■„
H
K
*>!
Eco FU
Hind III
Pst I
Sal I
Bam H I
Sma I
Eco R I
Hind III
Pst I
Sal I
Bam H I
Sma I
Eco R I
O
l“
o
z
m
ro
O
r~
O
z
m
Ol
1iL

Figure 17. Southern blot analysis of the hemagglutinating E. ooli
(A) Agarose gel (1.2%) showing restriction digests of the DNA.
Lanes: 1, pUC 9 digested with Hind III; 2, recombinant plasmid
from clone 2 digested with Hind III, Eco RI, and Sma I; 3, recom¬
binant plasmid from clone 5 digested with Hind III; 4, recombinant
plasmid from clone 7 digested with Hind III; 5, recombinant plasmid
from clone 5 digested with Hind III and Bam HI; 6, recombinant
plasmid from clone 7 digested with Hind III and Bam HI.
(B) Autoradiograph of DNA in panel A after Southern transfer and
hybridization with 32p_iabeled recombinant DNA from clone 7.

76

77
restricted with Hind III alone and Hind III together w'ith Bam HI
resulted in pUC 9 and an insert of 4,800 bp (Figure 17, panel A, lane
4), and pUC 9, insert of 2,800 and 2,000 bp (Figure 17, panel A, lane
6), respectively.
Hybridization of these transferred restricted DNAs demonstrated
that the clone 7 probe hybridized to pUC 9 and the common insert of
clones 5 and 7 but not to the insert of clone 2 (Figure 17, panel B).
Subcloning of Clone 5 for Autoagglutination and Hemagglutination
Clone 5 was found to agglutinate erythrocytes and autoagglutinate
while clone 7 was only able to agglutinate erythrocytes. Clone 5 has
an insert of 760 bp in addition to the common insert of 4,800 bp of
clone 7. This data suggested that the 760 bp insert might encode for
the autoagglutinating activity and the 4,800 bp fragment for the
hemagglutinating activity of clone 5. The recombinant plasmid of clone
5 was thus digested with Hind III to generate pUC 9 and inserts of
4,800 and 760 bp. Each insert band was isolated from the agarose gel
and ligated to Hind III cut pUC 9 and transformed into E. coli JM
109. The plasmids w'ere isolated from these transformants and digested
with restriction endonucleases. Subclones with different orientations
of the insert were obtained. Subclones of 760 bp inserts w'ere
designated clone 5.1 and 5.2 and the subclones of 4,800 bp inserts,
clone 5.3 and 5.4. Recombinant plasmids of clones 5.1 and 5.2 digested
wdth Hind III did result in pUC 9 and the 760 bp inserts (Figure 18,
lanes 2 and 3), and different patterns of restricted DNAs were seen
when digested with Sal I (Figure 18, lanes 6 and 7). Hind Ill-
restricted recombinant plasmids of clones 5.3 and 5.4 revealed pUC 9

78
and inserts of 4,800 bp (Figure 18, ianes 4 and 5), while Eco RI-
restricted recombinant plasmids showed different patterns (Figure 18,
lanes 8 and 9). Both clones 5.1 and 5.2 were able to autoagglutinate
when resuspended in PBS, pH 7.2, but could not agglutinate
erythrocytes. Clones 5.3 and 5.4 were both able to agglutinate
erythrocytes but did not autoagglutinate.
Western Blot Analysis of B. gingivalis Antigens Synthesized in
hemagglutinable E. coli
Upon Western blot analysis of clone 2, a protein antigen of
approximately 125 K and a smear of lower molecular weight were detected
using E. coli adsorbed anti-J3. ginglvalis antiserum but antigens
expressed in clones 5 and 7 were not detected by Western blot
analysis (described in Chapter Two). In an attempt to detect antigen
expression of DNA inserts in clones 5 and 7 and to achieve expression
of a more stable product from clone 2, the recombinant plasmids from
these clones were transformed into E. coli LC 137 IhtpRiAmTs) lonR 9
(Ts) lad Am) trp{ Am) pho( Am) rpsL supCi Ts) mal( Am) tsx::TnlO] kindly
provided by A. L. Goldberg. This bacterial strain has mutations in the
htpR and Ion genes, the products of which are involved in intracellular
protein degradation. However, this attempt was not successful since
the expressed antigen of clone 2 was still degraded and antigen
expression of clones 5 and 7 was not detected.
Identification of Native B. gingivalis Antigens
In order to determine the native B. gingivalis antigens which
clone 2 expressed, antisera against clone 2 were made in rabbits for

Figure 18. Agarose gel electrophoresis of recombinant plasmids
from clones 5.1, 5.2, 5.3, and 5.4.
Lanes: 1, DNA marker-Hind III digest of lambda DNA; 2-5,
recombinant plasmids from clones 5.1, 5.2, 5.3, and 5.4 digested
with Hind III; 6 and 7, recombinant plasmids from clones 5.1 and
5.2 digested with Sal I; 8 and 9, recombinant plasmids from clones
5.3 and 5.4 digested with Eco RI.


81
use as a probe in Western blot analysis. Pooled anti-clone 2 antiserum
had a titer of 1:16,000 against B. gingivalis whole cell antigen. This
antiserum was adsorbed exhaustively with E. coli JM 109 (pUC 9) until
the anti-FL coli titer was reduced from 1:50,000 to 1:10 in the E. coli
whole cell ELISA. The adsorbed antiserum, diluted to 1:200, was used
as a probe to detect antigens separated in a 12.5% SDS polyacrylamide
gel and transferred to a nitrocellulose sheet. As can be seen in
Figure 19, this antiserum reacted with 2 major bands of approximately
MWs 43,000 and 38,000 and 2 bands of MWs 32,000 and 30,000 in B.
gingivalis cell lysate antigen and the 125 K protein band of expressed
antigen in clone 2. Normal rabbit serum reacted to a common 40,000
molecular weight band of all the clones and E. coli JM 109 (pUC 9).
In order to prove that the B. gingivalis reactive polypeptides are
exclusively B. gingivalis proteins, the native B. gingivalis antigens
were reacted to E. coli adsorbed anti-clone 2 antiserum, B. gingivalis
cell lysate antigen and clone 2 whole cell antigen were again separated
in 12.5% SDS polyacrylamide gel. Upon transfer to a nitrocellulose
sheet, each was reacted with 1) E. coli adsorbed anti-clone 2 antiserum,
2) B. gingivalis adsorbed anti-clone 2 antiserum, and 3) antisera to
E. coli JM 109 harboring pUC 9 with an Eikenella corrodens DNA insert.
As can be seen in Figure 20, E. coli adsorbed anti-clone 2 reacted to B.
gingivalis cell lysate at 2 major bands of MWs 43,000 and 33,000, 2
bands of MWs 32,000 and 30,000 and 3 faint bands of higher molecular
weight of approximately 110,000, 90,000, and 75,000 daltons. This
adsorbed antiserum also reacted to a 125,000 MW band of expressed
antigen in clone 2. B. gingivalis adsorbed anti-clone 2 and anti-
E. coli JM 109 harboring pUC 9 with Eikenella DNA insert antisera did

Figure 19. Western blot analysis of native B. gingivalis antigens expressed by clone 2.
Lanes: 1, B. gingivalis cell lysate (40 nq); 2 to 7, whole cell samples of clones 2, 3,
4, 5, 7, and E. ooli JM 109 (pUC 9) as described in the methods.
(A) The blot was probed with E. coli-adsorbed anti-clone 2 antiserum.
(B) The blot was probed with normal rabbit serum.

83

Figure 20. Western blot analysis of native B. gingivalis antigens
expressed by clone 2.
Lanes: 1, B. gingivalis cell lysate (40 ug); 2, whole cell sample
of clone 2.
(A) The blot was probed with E. coli adsorbed anti-clone 2 antiserum.
(B) The blot was probed with B. gingivalis adsorbed anti-clone 2
anti serum.
(C) The blot was probed with antiserum against E. coli JM 109
harboring pUC 9 with an Eikenella DNA insert.

85
A
1 2
B
C
94 K
67K
43K
■ fctjjÉKSjy
30K
20K
14 K
fjrf

86
not react to B. gingivalis antigens or to the expressed antigen oi
clone 2 but reacted with E. coli antigens in clone 2.
To define the native B. gingivalis antigens which clones 5 and 7
expressed, antisera against clones 5 and 7 w^ere also made in rabbits
and had titers of 1:800 and 1:1,600 to B. gingivalis antigens. These
antisera exhaustively adsorbed with E. coli were used to identify the
reactive native B. gingivalis antigens. Antisera against clones 5 and 7
at the dilution of 1:5 and 1:10 were found to react with 2 bands of
approximately 43,000 and 38,000 daltons in B. gingivalis cell lysate
antigen preparation but did not react to the expressed clone 2 antigen
(Figure 21). This antiserum also reacted to a common band of
approximately 36,000 daltons of E. coli antigen in each clone and E.
coli JM 109 (pUC 9). Normal rabbit serum did not react to any B.
gingivalis antigens (Figure 21).
In order to determine if the anti-clones 2, 5, and 7 antisera were
reacting with the same B. gingivalis polypeptides or with different
peptides of similar migration rates, four samples of B. gingivalis
cell lysate antigens were separated in a 12.5% SDS polyacrylamide gel,
transferred to nitrocellulose paper and reacted with anti-clone 2
antiserum diluted 1:200, anti-clone 5 antiserum diluted 1:5, anti-clone
7 antiserum diluted 1:10, and a mixture of anti-clones 2, 5, arid 7
antisera at final concentrations of 1:200, 1:5, and 1:10, respectively.
Any differences in the pattern of reaction were undiscernable in these
4 blots (Figure 22).

Figure 21. Western blot analysis of native B. gingivalis antigen expressed by clone 7.
Lanes: 1, B. gingivalis cell lysate (40 ug); 2 to 5, whole cell samples of clones 2, 5,
7, and E. coli JM 109 (pUC 9) as described in the methods.
(A) The blot was probed with E. coli-adsorbed anti-clone 7 antiserum.
(B) The blot was probed with normal rabbit serum.

1
94 K
67K .
43K
30K
20 K .
14 K
5
s- ,:.t\ ' â– 
'M'W--"-
• < : .
É t ’Ü
. ■"■■■¿¿Sr '
'•i ? '
ía i i • > >
■ -■ .•
V
"5V â–  ;â– .
co
co

Figure 22. Western blot analysis of native B. gingivalis
antigens expressed by clones 2, 5, and 7. Forty ug of
B. gingivalis cell lysate was separated in a 12.5% SDS
polyacrylamide gel, transferred to a nitrocellulose sheet,
and probed with 1) E. coli adsorbed anti-clone 2 antiserum
diluted 1:200; 2) E. coli adsorbed anti-clone 5 antiserum
diluted 1:5, 3) E. coli adsorbed anti-clone 7 antiserum
diluted 1:10; and 4) a mixture of the above antisera.

90
12 3 4
__ ]
94 K
67 K
43K
30K
20 K
14 K _

91
Determination of The Relationship between the Clones 2, 5, and_7
expressed antigens
Although antisera against clones 2, 5, and 7 reacted to B.
gingivalis cell lysate at 2 major bands of 42,000 and 38,000 MWs
(Figure 23, lane 1 of panel A, B, and C), E. coli adsorbed anti-clone 2
antiserum also reacted to the 125 K protein band synthesized in clone 2
(Figure 23, panel A, lane 2). However, E. coli adsorbed anti-clone 5
and anti-clone 7 antisera did not react to this expressed antigen band
of clone 2 (Figure 23, lane 2 of panel B and C).
To further define the relationship of the epitopes of the
expressed antigen in clone 2 from that of clones 5 and 7, adsorption of
anti-clone 2 antiserum with several antigens was performed and each
adsorbed anti-clone 2 antiserum was tested for its titer to B.
gingivalis whole cell antigen by ELISA. These results were as shown
in Figure 24. The antibody titer to B. gingivalis of anti-clone 2
antiserum was removed in a dose response manner by adsorption with B.
gingivalis and clone 2 cells. Adsorption with E. coli JM 109 (pUC 9),
clone 5 or clone 7 did not reduce the antibody titer to B. gingivalis
of anti-clone 2 antiserum.
Hemagglutination Inhibition
The ability of antisera to B. gingivalis and hemagglutinable E.
coli to inhibit the hemagglutinating activity of B. gingivalis was
determ.-ned and is summarized in Table 4. Ail antisera inhibited B.
gingivalis hemagglutination at titers 4 to 8 times that of normal
rabbit sera.

Figure 23. Detection of B. gingivalis antigens synthesized by
clones 2, 5, and 7 as determined by Western blot analysis.
Lanes: 1, B. gingivalis cell lysate (40 ug); 2, whole cell
sample of clone 2.
(A) The blot was probed with E. coli adsorbed anti-clone 2
diluted 1:200.
(B) The blot was probed with E. coli adsorbed anti-clone 5
diluted 1:5.
(C) The blot was probed with E. coli adsorbed anti-clone 7
diluted 1:10.

92
94 K
67K
43 K
I
30K
20K
14 K

Figure 24. ELISA of anti-clone 2 antiserum adsorbed with
various numbers of cells of B. gingivalis ( o ), E. aoli
JM 109 harboring pUC 9 ( • ), clone 2(a), clone 5(a),
and clone 7(a). The adsorbed antisera were added to
B. gingivalis-coated microtiter plates and the assay was
performed as described in the methods.

OD 492nm

96
Table 4. Inhibition of hemagglutinating activity of B. gingivalis
by anti-hemagglutinating E. coli antisera.
Antiserum
Hemagglutination inhibition titer
Anti-B. gingivalis
unadsorbed
640
adsorbed with E. coli JM
109 (pUC 9)
640
Normal rabbit serum8
160
Anti-clone 2
320 - 640
Preimmune
80
Anti-clone 5
160
Preimmune
40
Anti-clone 7
160
Preimmune
40
8 Normal rabbit serum and preimmune sera titers are from each
particular group of rabbits.

97
The ability of each hemagglutinating clone to remove the hemagglu¬
tination inhibition activity of anti-5. gingivaJis antiserum was tested
and the results are shown in Table 5. Each clone partially removed
hemagglutination inhibition activity of anti-B. gingivalis antiserum.
Clones 2 and 7 decreased the hemagglutination inhibition titer of B.
gingivalis antiserum 2 to 4 fold. Adsorption of the antisera with
clone 5 cells reduced the titer 4 to 8 fold and a mixture of these
clones reduced the titer 8 fold. B. gingivalis itself reduced the
titer 16 fold and E. coli JM 109 (pUC 9) had no effect on the
inhibition activity of the antiserum.
Discussion
When antigen-expressing clones were surveyed for functional
activities, clones 2, 5, and 7 were able to agglutinate erythrocytes
whereas E. coli JM 109 (pUC 9) was not. The restriction maps and
Southern blot hybridization of these clones indicated that clone 2
cells contain a different Bacteroides DNA insert from clones 5 and 7.
Clone 5, which is also able to autoagglutinate, has a 760 bp DNA
fragment in addition to a 4,800 bp fragment in common with the clone 7
insert. Subcloning of these 2 fragments in different orientations
revealed that the 4,800 bp DNA encoded for the hemagglutinating
activity and the 760 bp DNA for the autoagglutinating activity. Both
fragments must contain a Bacteroides promoter since the subclones with
opposite orientations of the inserts still express functional proteins.
These data agree with the previous results described in Chapter Two
indicating that antigen expression of clones 5 and 7 is not stimulated
by IPTG. Unfortunately, B. gingivalis antigens expressed in clones 5

98
Table 5. Inhibition of hemagglutinating activity of B. gingivalis
by adsorbed anti-5, gingivalis antiserum
Antiserum
Hemagglutination inhibition titer
Anti-B. gingivalis
unadsorbed
640
adsorbed with
E. coli JM 109 (pUC 9)
640
adsorbed with
B. gingivalis
40
adsorbed with
clone 2
160 - 320
adsorbed with
clone 5
80 - 160
adsorbed with
clone 7
160 - 320
adsorbed with
clones 2, 5, and 7
80

99
and 7 could not be detected by Western blot analysis. Other techniques
such as in vitro transcription-translation and imraunoprecipitation
might be appropriate to detect the expressed antigens.
To date, the hemagglutinin of B. gingivalis strain 381 has been
partially purified in 2 different laboratories. The partially purified
hemagglutinin isolated by Inoshita et al (1986) separated by SDS-PAGE
demonstrated major protein bands of MWs 44 K, 37 K, and 24 K as well
as bands at 30 K and greater than 100 K. Okuda et al. (1986) also
purified a hemagglutinin, of which the SDS-PAGE pattern contained a
major protein band of 40 K in addition to several protein bands in
lesser amounts. A previous report by Naito et al. (1985) demonstrated
that monoclonal antibody directed against B. gingivalis hemagglutinin
(but not LPS or capsule), reacted with 40 K and 60 K bands of a
hemagglutinin preparation. However, these bands appear to be
approximately 40 K and 45 K, compared to the molecular weight standards
in their blot. The Fab fragments of this monoclonal antibody were
shown to inhibit the hemagglutinating activity of B. gingivalis. It
thus appears that the B. gingivalis hemagglutinin may contain at least
2 protein bands as detected by SDS-PAGE. However, the structure and
composition of the native molecule of B. gingivalis hemagglutinin is
still unknown.
In the studies described here, E. coli adsorbed rabbit-polyclonal
antibody against clone 2 was found to react with several bands in the
B. gingivalis cell lysate preparation separated by SDS-PAGE. The most
rapidly developing and strongest reaction appeared at 2 bands of 43 K
and 38 K. Two bands of 32 K and 30 K appeared later and 3 faint bands
of 110 K, 90 K, and 75 K sometimes were visible still later. This

100
strongly suggests that the B. gingivalis hemagglutinin is expressed in
clone 2. The DNA insert of clone 2 might encode for all four of the
polypeptides of 44 K, 38 K, 32 K, and 30 K or only a portion of some of
these polypeptides of B. gingivalis. E. coli JM 109 has a
nonsense suppressor tRNA, in which tRNA is able to suppress the
termination codon (UAG) at the end of the gene that uses this codon.
Therefore, the mRNA may be read through, resulting in the synthesis of
the intact protein band of 125 K which might also contain a 10 K
peptide of part of the p-galactosidase peptide. The 38 K polypeptide
might be part of the 44 K peptide since it has been shown that
monoclonal antibody reacts to 2 bands of similar molecular weights
(Naito et al., 1985). The two peptides could be generated from a
single gene by starting or terminating expression at different points.
It is also possible that the clone 2 insert encodes the long
polypeptides of approximately 105 K and 100 K and 90 K in B. gingivalis.
These long peptides might be generated from a single gene by starting
or stopping expression at different points. Once the cells of B.
gingivalis are disrupted as prepared for cell lysate sample, the long
polypeptides may be degraded into smaller peptides of 43 K, 38 K, 32 K,
and 30 K. Therefore, the high molecular weight band in the B. gingivalis
cell lysate to which antiserum against clone 2 reacted might be the
precursor of the smaller polypeptides.
E. coli adsorbed rabbit-polyclonal antibody against clones 5 and 7
also reacted with 2 bands of 43 K and 38 K, but barely reacted with the
higher bands of 110 K, 90 K, and 75 K and did not react w'ith the bands
of 32 K and 30 K. Thus, these clones and clone 2 contain nonhomologous
DNA inserts and express different antigenic epitopes but all function

101
as hemagglutinins. Two possibilities that may explain this phenomenon
are (1) different Bacteroides components migrated in SDS-PAGE the same
distance. This however, appears unlikely. To resolve these bands, a
technique of two-dimensional gel electrophoresis must be performed. (2)
each cloned insert encodes a different portion of the same polypeptide
of B. gingivalis and each portion with different antigenic epitopes
can function as a hemagglutination.
In the latter case, the clone 7 insert contains a Bacteroides
promoter but the clone 2 insert does not. The 43 K and 38 K polypeptides
might be generated from a single gene by starting (or terminating)
expression at different points. The clone 7 insert or part of the
insert would code for the N-terminus of the 43 K polypeptide and end
some where at the N-terminus of the 38 K polypeptide. The clone 2
insert may code for the same C-terminus of the 43 K and 38 K
polypeptides and might contain the structural gene for other peptide
(32 K and 30 K) to u'hich antiserum against clone 2 reacts.
Hemagglutination inhibition by antiserum against each clone might
result from spatial interference, i.e., Fab portions of antibody
molecules might bind to other antigens of B. gingivalis which are close
to, but are not actually hemagglutinin antigens. Therefore, they can
hinder hemagglutinins from binding erythrocytes. In order to exclude
this possibility, Fab fragments should be prepared from the IgG of
these antisera and tested for their ability to inhibit hemagglutination
of B. gingivalis. However, the ability of each clone to partially
remove the hemagglutination inhibition activity of anti-B. gingivalis
antiserum indicates that there are more than one antigenic determinant
which functions as a hemagglutinin. It may be either the case of 2

102
functional epitopes of the same polypeptide or the case of 2 different
hemagglutinin components.

CHAPTER FOUR
CONCLUSION
The hemagglutinating activity of B. gingivalis has been studied as
a parameter that affects the adherence of this organism in the
periodontal pocket since B. gingivalis possesses fimbriae (Okuda and
Takazoe, 1974; Slots and Gibbons, 1978) and many fimbriae of other
bacterial species have hemagglutinating activity as well as the ability
to adhere to a variety of host cells (Pearce and Buchanan, 1980). It
has been recently reported that sera from patients with adult
periodontitis possess high antibody levels to the B. gingivalis
hemagglutinin. It is thus suggested that the adhesive surface
structures such as hemagglutinin participate in B. gingivalis
colonization and antigenic stimulation of the host (Naito et al.,
1987). Hemagglutinating activity has also been shown to be a unique
characteristic of oral strains of B. asaccharolyticus (presently B.
gingivalis). Nonoral strains of B. asaccharolyticus do not have
hemagglutinating activity (Slots and Genco, 1979). Components of
bacteria which mediate attachment to host tissues include surface
structures such as fimbriae, capsuiar material, lipopolysaccharide, and
membrane-associated extracellular vesicles (Slots and Genco, 1984).
Fimbrial preparations from B. gingivalis 381 have been found to possess
strong hemagglutinating activity but LPS or polysaccharide apparently
do not (Okuda et al., 1981). However, Boyd and McBride (1984) have
103

104
reported that an outer membrane preparation of B. gingivalis W12
mediates hemagglutination but does not contain fimbriae-like structures.
In addition, Yoshimura et al. (1984) purified a novel type of fimbriae
from B. gingivalis 381 which did not show hemagglutinating activity.
Recently, Okuda et al. (1986) have demonstrated that the monoclonal
antibody against B. gingivalis, which reacted to partially purified
hemagglutinin but not to LPS or capsule, bound to fibrillar structures
of B. gingivalis. It is thus unclear whether the B. gingivalis
hemagglutinin is present on fimbriae or not.
The hemagglutinin of B. gingivalis has not yet been completely
purified and characterized. However, a few investigations have
reported the isolation of hemagglutinin activity from B. gingivalis.
Boyd and McBride (1984) prepared an outer membrane component containing
hemagglutinating activity from B. gingivalis W12. This preparation
contained three major proteins with molecular weights of 69,000,
41,500, and 22,000. Inoshita et al. (1986) isolated hemagglutinating
activity from culture supernatants of B. gingivalis 381. The isolated
hemagglutinin component contains three major proteins with molecular
weights of 24,000, 37,000, and 44,000. Okuda et al. (1986) also
purified a hemagglutinin of B. gingivalis 381 from culture supernatant
which appears to have vesicle or tubelike structures and is comprised
mainly of a 40,000 molecular-weight protein. Their recent report
indicated that sera from most patients with adult periodontitis reacts
to the hemagglutination antigen at 43,000 and 57,000 molecular weights
(Naito et al., 1987).
This report describes the cloning of B. gingivalis chromosomal DNA
in E. coli which then enables the E. coli host to bind erythrocytes,

105
the reaction of antisera against hemagglutinable E. coli with
B. gingivalis antigens which have been previously reported to be the
major antigens in partially purified hemagglutinin, and the ability of
each hemagglutinable E. coli to partially remove the hemagglutination
activity of anti-B. gingivalis antiserum. These data strongly suggest
that I have cloned B. gingivalis hemagglutinin genes into E. coli.
In addition, the results indicate that two different B. gingivalis
DNA sequences express different antigenic epitopes, both of w'hich
function as hemagglutinins. To determine whether these cloned DNA
fragments are adjacent in Bacteroides chromosomal DNA (i.e., contained
in one gene) or not, the following experiments should be performed.
1. Hybridization of restricted B. gingivalis chromosomal DNA
wúth DNA inserts from clones 2 and 7. Each DNA insert should hybridize
to the same chromosomal DNA fragment if these inserts are contiguous on
the chromosomal DNA. Appropriate restriction enzymes should be used to
digest chromosomal DNA completely in order to generate fragments w'hich
are not cut at a site between these inserts. For partial digestion,
conditions of digestion of the DNA must be adjusted in order to
maximize the possibility of obtaining similar hybridization patterns
from both inserts if the sequences are contiguous.
2. Chromosome walking technique. This protocol would be
accomplished by using the insert from each clone to isolate the
adjacent DNA fragment. For example, the DNA insert of clone 7 would be
used as a probe to identify other recombinant plasmids in the DNA
library that overlap with clone 7 DNA. These new recombinant plasmids
will contain DNA sequences which extend on one side or the other from
the fragment carried in clone 7. The direction of extension can be

106
determined by producing a restriction map of each fragment. This
process must be repeated until reaching the overlap region of clone 2
DNA.
3. Isolation of the clones that contain inserts from both clones
2 and 7. This may be achieved by using the inserts from clones 2 and 7
to screen the DNA library by DNA hybridization. The clones that
contain homologous sequences to both clones 2 and 7 inserts will be
analyzed for their DNA inserts.
4. Two-dimensional gel electrophoresis. The possibility of
different hemagglutinin components migrating the same distance in
SDS-PAGE can be explored by analysis of B. gingivalis antigens in two
dimensional gel electrophoresis and probing with the antisera against
each clone.
5. Affinity purification of the polypeptide reactive with anti¬
clone 2 antiserum and determination of reactivity with anti-clone 7
antiserm. B. gingivalis antigens synthesized by clone 2 could be
purified by immunoaffinity chromatography employing immobilized
polyclonal antiserum against clone 2. The Bacteroides antigens which
antiserum against clone 2 recognize will be bound to the column and
will be eluted with a pH gradient. The isolated antigens could then be
tested for reactivity with the antiserum against clone 7.
In addition, the isolated antigens should be tested for
hemagglutination activity and analyzed in SDS-PAGE for determination of
the molecular weight. The isolation of B. gingivalis antigens which
possess hemagglutination activity should confirm that these cloned DNAs
are hemagglutinin genes of B. gingivalis. The peptide maps or peptide
fingerprints in two-dimensional gel electrophoresis of these isolated

107
proteins will definitely confirm that they are the same or different
proteins and thus determine if one or more components of B. gingivalis
mediate hemagglutination.
In order to define the genes which encode the hemagglutinin(s),
subcloning should be performed. The DNA of functional subclones with
the smallest insert could then be sequenced. The predicted amino acid
sequence will provide information such as the isoelectric point (p1)
which would facilitate the purification of the protein.
Finally, the evidence which proves that hemagglutinin genes have
been cloned must be generated by producing B. gingivalis mutants which
lack hemagglutinating activity. If a transformation system is developed
for B. gingivalis, it would then be possible to mutageriize the cloned
sequence arid introduce the mutated gene into B. gingivalis, thereby
knocking out the hemagglutinating ability of the cells. B. gingivalis
mutants devoid of hemagglutinating activity are necessary for in vivo
studies to establish the significance of hemagglutination in
pathogenesis. The cloning of hemagglutinin(s) which are suspected to
mediate attachment to host tissues provides a means to define the native
structures of B. gingivalis responsible for this activity. The cloned
hemagglutinin genes are also good candidates for DNA probes for the
rapid identification of B. gingivalis in clinical samples. Ultimately,
these cloned genes may facilitate the production of a vaccine which
prevents the colonization of B. gingivalis in the gingival sulcus and
possibly the prevention of periodontal disease.

LITERATURE CITED
Bom-van Noorloos, A. A., C. A. Schipper, T. J. M. van Steenbergen, J.
de Graff, and E. H. Burger. 1986. Bacteroides gingivalis
activates mouse spleen cells to produce a factor that stimulates
resorptive activity of osteoclasts in vitro. J. Periodont. Res.
21:440-444.
Boyd, J. and B. C. McBride. 1984. Fractionation of hemagglutinating
and bacterial binding adhesins of Bacteroides gingivalis. Infect.
Immun. 45:403-409.
Burnette, W. N. 1981. "Western blotting": Electrophoretic transfer of
proteins from sodium dodec-yl sulfate-polyacrylamide gels to
radiographic detection with antibody and radioiodinated protein A.
Anal. Biochem. 1 12:195-203.
Carlsson, J., B. F. Herrmann, J. F. Hofling, and G. K. Sundqvist.
1984a. Degradation of the human proteinase inhibitors alpha-1-
antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis.
Infect. Immun. 43:644-648.
Carlsson, J., J. F. Hofling, and G. K. Sundqvist. 1984a. Degradation
of albumin, haemopexin, haptoglobin, and transferrin by black
pigmented Bacteroides species. J. Med. Microbiol. 18:39-46.
Castello, A. H., J. O. Cisar, P. E. Kolenbrander, and 0. Gabriel.
1979. Neuraminadase-dependent hemagglutination of human
erythrocytes by human strains of Actinomyces viscosus and
Actinomyces naeslundii. Infect. Immun. 26:563-572.
Charette, M. F., G. W. Henderson, and A. Markovitz. 1981. ATP
hydrolysis-dependent protease activity of the Ion (cap R) protein
of Escherichia coli K 12. Proc. Natl. Acad. Sci. USA
78:4728-4732.
Chung, C. H. and A. L. Goldberg. 1981. The product of Ion (capR) gene
in Escherichia coli is the ATP-independent protease, protein La.
Proc. Natl. Acad. Sci. USA 78:4931-4935.
Cisar, J. 0. 1982. Coaggregation reactions between oral bacteria:
Studies of specific cell-to-cell adherence mediated by microbial
lectins, pp. 121-131. In: R. J. Genco, and S. E. Mergenhagen
(eds.). Host-parasite interactions in periodontal disease.
American Society for Microbiology, Washington, D.C.
Cisar, J. O., E. L. Barsumian, S. H. Curl, A. E. Vatter, A. E.
Sandberg, and R. P. Siraganian. 1981. Detection and Localization
of a lectin on Sctinomyces viscosus T 14 V by monoclonal
antibodies. J. Immunol. 127:1318-1322.
108

109
Cisar, J. O., P. E. Kolenbrander, and F. C. Melinite. 197S.
Specificity of coaggregation reactions between human oral
streptococci and strain of Actinomyces viscosus or Actinomyces
naeslundii. Infect. Immun. 24:742-752.
Clark, W. B., L. L. Bammann, and R. J. Gibbons. 1978. Comparative
estimates of bacterial affinities and adsorption sites on
hydroxyapatite surfaces. Infect. Immun. 19:846-853.
Coykendall, A. L., F. S. Kaczmarek, and J. Slots. 1980. Genetic
heterogeneity in Bacteroides asaccharolyticus (Holdernan and Moure,
1970). Finegold and Barnes, 1977 (approved lists, 1980) and
proposal of Bacteroides gingivaiis sp. nov. and Bacteroides
macacae (Slots and Genco) comb. nov. Int. J. Sys. Bacteriol. 30:
559-564.
Crawford, A. C. R., S. S. Socransky, E. Smith, and R. Phillips. 1977.
Pathogenicity testing of oral isolates in gnotobiotic rats. J.
Dent. Res. 56: (Special Issue B). Abstract # 275.
De Franco, A. L. and D. E. Koshland, Jr. 1981. Molecular cloning of
chemotoxins genes and overproduction of gene products in the
bacterial sensing system. J. Bacteriol. 147: 390-400.
Ellen, R. P., E. D. Fillery, K. H. Chan, and D. A. Grove. 1980.
Sialidase-enhanced lectin-like mechanism for Actinomyces
naeslundii hemagglutination. Infect. Immun. 27:335-343.
Engleberg, N. C., D. J. Drutz, and B. I. Eisenstein. 1984a. Cloning
and expression of Legionella pneumophila antigens in Escherichia
coli. Infect. Immun. 44:222-227.
Engleberg, N. C., E. Pearlman, and B. I. Eisenstein. 1984b. Legionella
pneumophila surface antigens cloned and expressed in Escherichia
coli are translocated to host cell surface and interact with
specific anti-Legionella antibodies. J. Bacteriol. 160:199-203.
Fairbanks, G., T. L. Steck, and D. F. H. Wallach. 1971.
Electrophoretic analysis of the major polypeptides of the human
erythrocyte membrane. Biochemistry 10:2602-2616.
Frank, R. and J. C. Voegel. 1978. Bacterial bone resorption in
advanced cases of human periodontitis. J. Periodont. Res. 13:
251-261.
Gibbons, R. J. 1984. Adherent interactions which may affect microbial
ecology in the mouth. J. Dent. Res. 63:378-385.
Gibbons, R. J. and I. Etherden. 1983. Comparative hydrophobicities of
oral bacteria and their adherence to salivary pellicles. Infect.
Immun. 41:1190-1196.
Gibbons, R. J. and J. Van Houte. 1975. Bacterial adherence in oral
microbiol ecology. Annu. Rev. Microbiol. 29:19-44.

110
Gibbons, R. J. and I. Etherden. 1983. Comparative hydrophobicities
of oral bacteria and their adherence to salivary pellicles.
Infect. Iramun. 41:1190— 1196.
Gibbons, R. J. and J. Van Houte. 1980. Bacterial adherence and the
formation of dental plaques, pp. 61-104. In: E. H. Beachey
(ed.), Bacterial Adherence (Receptors and recognition. Series B,
Vol. 6).
Gibbons, R. J., I. Etherden, and Z. Skobe. 1983. Association of
fimbriae with the hydrophobicitv of Streptococcus sanguis FC-1 and
adherence to salivary pellicles. Infect. Immuri. 41:414-417.
Goldberg, S. L. and J. R. Murphy. 1984. Molecular cloning of the
hemolysin determinant from Vibrio cholerae El Tor. J. Bacteriol.
160:239-244.
Grenier, D. and D. Mayrand. 1987. Functional characterization of
extracellular vesicles produced by Bacteroides gingivalis.
Infect. Immun. 55:111-117.
Health, J. K., S. J. Atkinson, R. M. Hembry, J. J. Reynolds, and M. C.
Meikle. 1987. Bacterial antigens induce collagenase and
prostaglandin E2 synthesis in human gingivalis fibroblasts
through a primary effect on circulating mononuclear cells. Infect.
Immun. 55:2148-2154.
Horton, J. E., J. J. Oppenheim, and S. E. Mergenhagen. 1974. A role
for cell-mediated immunity in the pathogenesis of periodontal
disease. J. Periodontal. 45:351-360.
Horton, J. E., L. G. Raisz, H. A. Simmons, J. J. Oppenheim, and S. E.
Mergenhagen. 1972. Bone resorbing activity in supernatant fluid
from cultured human peripheral blood leukocytes. Science 177:
793-795.
Inoshita, E., A. Amano, T. Hanioka, H. Tamagawa, S. Shizukushi, and A.
Tsunemitsu. 1986. Isolation and some properties of exohernagglu-
tinin from the culture medium of Bacteroides gingivalis 381.
Infect. Immun. 52:421-427.
Ish-Horowicz, D. and J. F. Burke. 1981. Rapid and efficient cosmid
cloning. Nucleic Acids Res. 9:2989-2998.
Ivanyi, L. and T. Lehner. 1970. Stimulation of lymphocyte trans¬
formation by bacterial antigens in patients with periodontal
disease. Arch. Oral. Biol. 15:1089-1096.
Jayawardene, A. and M. Goldner. 1977. Reagin-like activity of serum
in human periodontal disease. Infect. Immun. 15:665-667.
Kagan, J. M. 1980. Local Immunity to Bacteroides gingivalis in
Periodontal Disease. J. Dent. Res. 59 DL1750-1756.

Ill
Kilian, M. 1981. Degradation of immunoglobulins Al, A2, and G by
suspected principal periodontal pathogens. Infect. Irnmun. 34:
757-7 65.
Klainfeldt, A. 1986. Degradation of bovine articular cartilage
proteoglycans in vitro. Seand. J. Rheumatol. 15:297-301.
Kollenbrander, P. E. and B. L. Willaims. 1981. Lactose-reversible
coaggregation between oral actinomycetes and Streptococcus
sanguis. Infect. Irnmun. 33:95-102.
Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (London) 227:
680-685.
Lantz, M. S., R. W. Rowland, L. M. Switalski, arid M. Hook. 1986.
Interactions of Bacteroides gingivalis with fibrinogen. Infect.
Irnmun. 54:654-658.
Laughon, D. E., S. A. Syed, and W. J. Loesche. 1982. API ZYM system
for identification of Bacteroides spp., Capnocytophaga spp.,
and Spirochetes of oral origin. J. Clin. Microbiol. 15:97-102.
Layman, D. L. and D. L. Diedrich. 1987. Growth inhibitory effects of
endotoxins from Bacteroides gingivalis and intermedius on human
gingival fibroblasts in vitro. J. Periodontol. 58:387-392.
Leong, D. L., C. I. Hoover, J. R. Winkler, R. H. Kramer, and P. A.
Murray. 1985. Bacterial attachment to a basement-like matrix in
vitro (Abstract). J. Dent. Res. 64:330.
Lindhe, J. and S. S. Socransky. 1979. Chemotaxis and vascular
permeability produced by human periodontopathic bacteria. J.
Periodont. Res. 14:138-146.
Loesche, W. J. and S. A. Syed. 1978. Bacteriology of human
experimental gingivitis: Effect of plaque and gingivitis score.
Infect. Irnmun. 21:830-839.
Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning:
a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.
Matsumura, P., J. J. Rydel, R. Linzmeier, and D. Vacante. 1984.
Overexpression and sequence of the Escherichia coli che Y
gene and biochemical activities of the che Y protein. J.
Bacteriol. 160:36-41.
Mayrand, D. and D. Grenier. 1985. Detection of collagenase activity
in oral bacteria. Can. J. Microbiol. 31:134-138.
Mayrand, D. and B. C. McBride. 1980. Ecological relationships of
bacteria involved in a simple, mixed anaerobic infection. Infect.
Irnmun. 27:44-50.

112
Mayrand, D. B. C. McBride, T. Edwards, and S. Jensen. 1980. Charac¬
terization of Bacteroides asaccharolyticus and B. melaninogenecus
oral isolates. Can. J. Microbiol. 26:1178-1183.
McKee, A. S., A. S. McDermid, A. Baskerville, A. B. Dowsett, D. C.
Ellwood, and P. D. Marsh. 1986. Effect of hemin on the
physiology and virulence of Bacteroides gingivalis W50. Infect.
Immun. 52(2):349-355.
Meyer, T. F., N. Mlawer, and M. So. 1982. Pilus expression in Neisseria
gonorrhoeae involves chromosomal rearrangement. Cell 30:45-52
Mooney, J. J. and B. H. Waksman. 1970. Activation of normal rabbit
macrophage monolayers by supernatants of antigen stimulated
lymphocytes. J. Immunol. 105:1138-1145.
Mouton, C., P. G. Hammond, J. Slots, and R. J. Genco. 1981. Serum
Antibodies to ORal Bacteroides asaccharolyticus (Bacteroides
gingivalis) :Relation to Age and Periodontal Disease. Infect. Immun.
31:182-192.
Nair, B. C., W. R. Mayberry, R. Dziak, P. B. Chen, M. J. Levine, and
E. Hausmann. 1982. Biological effects of a purified lipopolysac-
charide from Bacteroides gingivalis. J. Periodontal Res. 18:40-49.
Naito, Y., K. Okuda, T. Kato, and I. Takazoe. 1985. Monoclonal
antibodies against surface antigens of Bacteroides gingivalis.
Infect. Immun. 50:231-235.
Naito, Y., K. Okuda, and I. Takazoe. 1984. Immunoglobulin G response
to subgingival gram-negative bacteria in human subjects. Infect.
Immun. 45:47-51.
Naito, Y., K. Okuda, and I. Takazoe. 1987. Detection of specific
antibody in adult human periodontitis sera to surface antigens of
Bacteroides gingivalis. Infect. Immun. 55(3):832-834.
Nakamura, T., S. Fujimura, N. Obata, and N. Yamazaki. 1981.
Bacteriocin-like substance (melaninocin) from oral 4Bacteroides
melaninogenicus. Infect. Immun. 31:28-32.
Nakamura, T., Y. Suginaka, N. Obata, N. Yamazaki, and I. Takazoe.
1978. Growth inhibition of Streptococcus mutans by the black
pigmented (Haematin) of Bacteroides melaninogenicus. Arch. Oral.
Biol. 23:593-595.
Nicosia, A., A. Bartoloni, M. Perugini, and R. Rappuoli. 1987.
Expression and immunological properties of the five subunits of
pertussis toxin. Infect. Immun. 55:963-967.
Nilsson, T., J. Carlsson, and G. Sundqvist. 1985. Inactivation of key-
factors of the plasma proteinase cascade systems byr Bacteroides
gingivalis. Infect. Immun. 50:467-471.

113
Nisengard, R. J. 1977. The role of immunology in periodontal disease.
J. Periodont. 48:505-516.
Okuda, K., A. Yamanoto, Y. Naito, I. Takazoe, J. Slots, and R. J.
Genco. 1986. Purification and properties of hemagglutinin from
culture supernatant of Baeteroides gingivalis. Infect. Immun. 55:
659-665.
Okuda, K., K. Yanagi, and I. Takazoe. 1978. Complement activation by
Propionibacterium acnés and Baeteroides melaninogenicus.
Arch. Oral Biol. 23:911-915.
Okuda, K., J. Slots, and R. J. Genco. 1981. Baeteroides gingivalis,
Baeteroides asaccharolyticus and Bacteriudes nekabubigebucys
subspecies: cell surface morphology and adherence to erythrocytes
and human buccal epithelial cells. Curr. Microbiol. 6:7-12.
Okuda, K. and I. Takazoe. 1974. Haemagglutinating activity of
Baeteroides melaninogenicus. Archs. Oral Biol. 19:415-416.
Oliver, D. 1985. Protein secretion in Escherichia coli. Annu. Rev.
Microbiol. 39:615-648.
Olsson-Wennstrom, A., J. L. Wennstrom, S. E. Mergenhagen, and R. P.
Siraganian. 1978. The mechanism of basophil histamine release
in patients with periodontal disease. Clin. Exp. Immunol. 33:166-
173.
Patters, M. R., P. Chen, J. McKenna, and R. J. Genco. 1980.
Lymphopro-liferative response to oral bacteria in humans with
varying severities of periodontal disease. Infect. Immun. 28:777-
784.
Patters, M. R., R. J. Genco, M. J. Reed, and P. A. Mashimo. 1976.
Blastogenic response of human lymphocytes to oral bacterial
antigens: Comparison of individuals with periodontal disease to
normal and edentulous patients. Infect. Immun. 14:1213-1220.
Patters, M. R., N. Sedransk, and R. J. Genco. 1979. The
lymphoproliferative response during human experimental gingivitis.
J. Periodont. Res. 14:269-278.
Pearce, W. A. and T. M. Buchanan. 1980. Structure and cell membrane¬
binding properties of bacterial fimbriae, pp. 289-344. In: E. H.
Beachey (ed.), Receptors and recognition series B, Vol. 6,
bacterial adherence. Chapman and Hall, London.
Pearson, G. P. N. and J. J. Mekalanos. 1982. Molecular cloning of
Vibrio cholerae enterotoxin genes in Escherichia coli K-12.
Proc. Natl. Acad. Sci. USA 79:2976-2980.
Perbal, B. 1984. A practical guide to molecular cloning. John Wiley
& Sons, Inc., N.Y.

114
Robertson, P. B., M. Lants, P. T. Marucha, K. S. Kornman, C. L.
Trummel, and S. C. Molt. 1982. Coilagenolytic activé
associated with Bacteroides species and Actinobacillus
actinomycetemcomitans. J. Periodontal Res. 17:275-283.
Schwarz, S., R. P. Ellen, and D. A. Grove. 1987. Bacteroides
gingivalis-actinomyces viscosus cohesive interactions as measured
by a quantitative binding assay. Infect. Immun. 55:2391-2397.
Silhavy, T. J., M. L. Berman, and L. W. Enquist. 1984. Experiments
with gene fusions. Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.
Singer, R. E. and B. A. Buckner. 1981. Butyrate and proprionate:
Important components of toxic dental plaque extracts. Infect.
Immun. 32:458-463.
Slots, J. 1977. The predominant cultivable microflora of advanced
periodontitis. Scand. J. Dent. Res. 85:114-121.
Slots, J. 1979. Subgingival microflora and periodontal disease. J.
Clin. Periodontal. 6:351-382.
Slots, J. 1981. Enzymatic characterization of some oral and nonoral
Gram-negative bacteria with the API ZYM system. J. Clin.
Microbiol. 14:288-294.
Slots, J. 1982. Importance of black pigmented Bacteroides in
periodontal disease, pp. 27-45. In: R. J. Genco and S. E.
Mergenhagen (eds.), Host-parasite interactions in periodontal
diseases. American Society for Microbiology, Washington, D.C.
Slots, J., L. Bragd, M. Wikstrom, and G. Dahlen. 1986. The occurrence
of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis
and Bacteroides intermedius in destructive periodontal disease in
adults. J. Clin. Periodontal. 13:570-577.
Slots, J. and R. J. Genco. 1979. Direct hemagglutination technique
for differentiating Bacteroides asaccharolyticus oral strains
from non oral strains. J. Clin. Microbiol. 10:371-373.
Slots, J. and R. J. Genco. 1984. Black-pigmented Bacteroides
species, Capnocytophaga species, and Actinobacillus
actinomycetemcomitans in human periodontal disease: Virulence
factors in colonization, survival, and tissue destruction. J.
Dent. Res. 63:412-421.
Slots, J. and R. J. Gibbons. 1978. Attachment of Bacteroides
melaninogenicus subsp. asaccharolyticus to oral surfaces and
its possible role in colonization of the mouth and of periodontal
pockets. Infect. Immun. 19:254-264.

115
Slots, J. and E. Hausmann. 1979. Longitudinal study of
experimentally induced periodontal disease in Macaca arctoides:
relationship between microflora and alveolar bone loss. Infect.
Immu . 23:260-269.
Socransky, S. S. 1977. Microbiology of Periodontal Disease -Present
status and Future considerations. J. Periodontal. 48:497-504.
Southern, E. M. 1975. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-
517.
Spiegel, C. A., S. E. Hayduk, G. E. Minah, and G. N. Krywolap. 1979.
Black-pigmented Dacteroides from clinically characterized
periodontal sites. J. Periodorit. Res. 14:376-382.
Stamm, L. V., J. D. Folds, and P. J. Bassford, Jr. 1982. Expression
of Treponema pallidum antigens in Escherichia coli K12. Infect.
Imrriun. 36:1238-1241.
Sundqvist, G., G. D. Bloom, K. Enberg, and E. Johansson. 1982.
Phagocytosis of Bacteroides gingivaiis in vitro by human
neutrophils. J. Periodont. Res. 17:113-121.
Sundqvist, G., J. Carlsson, B. Herrmann, and A. Tarnvik. 1985.
Degradation of human immunoglobulin G and M and complement factors
C3 and C5 by black pigmented Bacteroides. J. Med. Microbiol. 19:
85-94.
Sundqvist, G. and E. Johansson. 1980. Neutrophil chemmotaxis induced
by anaerobic bacteria isolated from necrotic dental pulps. Scand.
J. Dent. Res. 88:113-121.
Sundqvist, G. and E. Johansson. 1982. Bactericidal effect of pooled
human serum on Bacteroides mulaninogeicus, Bacteroides
asaccharolyticus, and Actiriohacillus actinomycetemcomitans.
Scand. J. Dent. Res. 90:29-36.
Sveen, K. 1977a. Rabbit polymorphonuclear leukocyte migration in vitro
in response to lipopolvsaccharides from Bacteroides, Fusobacterium,
and Veillonella. Acta Pathol. Microbiol. Scand. (B) 85:374-380.
Sveen, K. 1977b. Rabbit polymorphonuclear leukocyte migration in vivo
in response to lipopolysaecharides from Bacteroides, Fusobacterium,
and Veillonella. Acta Pathol. Microbiol. Scand. (B)
85:381-387.
Takazoe, I., T. Nakamura, and K. Okuda. 1984. Colonization of the
Subgingival area by Bacteroides gingivaiis. J. Dent. Res. 63:
422-426.
Tanner, A. C. R., C. Haffer, G. T. Bratthal, R. A. Viscontii, and S. S.
Socransky. 1977. A study of the bacteria associated with
advancing periodontitis in man. J. Clin. Periodontal. 6:278-307.

116
Tolo, K., K. Schenck, and J. R. Johansen. 1982. Activity of Human
Serum Immunoglobulins to Seven Anaerobic Oral Bacteria before and
after Periodontal Treatment. J. Periodont. Res. 17:481-483.
Tonzetich, J. and B. C. McBride. 1981. Characterization of volatile
sulphur production by pathogenic and . on-pathogenic strains of
oral Bacteroides. Arch. Oral. Biol. 26:963-965.
Tsutsui, H., T. Kinouchi, Y. W altano, and Y. Ohnishi. 1987.
Purification and characterization of a protease from Bacteroides
gingivalis. 381. Infect. Immun. 55:420-427.
Van Steenbergen, T. J. M., P. Kastelein, J. J. A. Touw. and J. De
Graaf. 1982. Virulence of black-pigmented Bacteroides strains
from periodontal pockets and other sites in experimentally induced
skin lesions in mice. J. Periodont. Res. 17:41-49.
Vasil, M. L., C. Chamberlain, and C. C. R. Grant. 1986. Molecular
studies of Pseudomonas erotoxin A gene. Infect. Immun. 52:538-
548.
Vieira, J. and J. Messing. 1982. The pUC plasmids, an M13 mp 7-
derived system for insertion mutagenesis and sequencing with
synthetic universal primers. Gene 19:259-268.
Vodkin, M. H. and S. H. Leppla. 1983. Cloning of the protective
antigen gene of Bacillus anthracis. Cell 34:693-697.
Wahl, S. M. 1982. Mononuclear cell-mediated alteraetions in
connective tissue, pp. 225-234. In: R. J. Genco and S. E.
Mergenhagen (eds.) Host-parasite interactions in periodontal
disease. American Society of Microbiology, Washington, D.C.
Wahl, G. M., M. Stern, and G. R. Stark. 1979. Efficient transfer of
large DNA fragments from agarose gels to diazobenzyloxy-methvl-
paper and rapid hybridization by using dextran sulphate. Proc.
Natl. Acad. Sci. USA 76:3683-3687.
Wahl, L. M., S. M. Wahl, S. E. Mergenhagen, and G. R. Martin. 1975.
Collagenase production by lymphokine activated macrophages.
Science 187:261-263.
Walker, J. A., R. L. Allen, P. Falmagne, and M. K. Johnson. 1987.
Molecular cloning, characterization, and complete nucleotide
sequence of the gene for pneumolysin, the sulfpydryl-activated
toxin of Streptococcus pneumoniae. Infect. Immun. 55:1184-
1189.
Waxman, L. and A. L. Goldberg. 1982. Protease La from Escherichia
coli hydrolyzes ATP and proteins in a linked fashion. Proc.
Natl. Acad. Sci. USA 79:4883-4887.

117
White, D. and D. Mayrand. 1981. Association of oral Bacteroides
with gingivitis and adult periodontitis. J. Periodcnt. Res. 11:
1-18.
Wikstrom. M. B.. G. Dahlen, and A. Linde. 1983. Fibrinogeriolytic and
fibrinolytic activity in oral microorganisms. J. Clin. Microbiol.
17:759-767.
Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13
phage cloning vectors and host strains: nucleotide sequences of
the Ml3 mp 18 and pUC 9 vectors. Gene 33:103-119.
Yoshimura, F., K. Takahashi, Y. Nodosaka, and T. Suzuki. 1984.
Purification and characterization of a novel type of fimbriae from
the oral anaerobe Bacteroides gingivalis. J. Bacteriol. 160:
949-957.
Zambón, J. J., H. S. Reynolds, and J. Slots. 1981. Black pigmented
Bacteroides spp. in the human oral cavity. Infect. Immun. 32:198-
203.
Zhu, J. W. Kempenaers, D. Van der Straeten, R. Contreras, and W. Fiers.
1985. A method for fast and pure DNA elution form agarose gels by
centrifugal filtration. Biotech. 3:1014-1016.

BIOGRAPHICAL SKETCH
Somying was born to Sanguan and Chaufa Juijaitrong on December 2,
1953. in Tahmuarig, Kanchariaburi, Thailand. Sornyirtg is married to
Sorrithep Tumwasorn and has 2 sons, Pattarawuth and Nattapol.
She lived in Tahrnuartg until 1969 when she went to Triem Udornsuksa
School in Bangkok. She entered Kasetsart University in June, 1971 and
was supported by the John F. Kennedy Foundation until she received a
Bachelor of Science (with honours) in food science and technology in
May, 1975. Upon graduation, she received a scholarship from the
University Development Commission to persue a master's degree in
microbiology at Kasetsart University. After graduation in May, 1977,
she joined the Department of Microbiology, Faculty of Medicine,
Chulalongkorn University as an instructor and was appointed as an
Assistant Professor in 1980.
In August, 1983, Somying entered the Graduate School of the
University of Florida in the Department of Immunology and Medical
Microbiology. Her Ph.D. program has been arranged and supported in
part by the Fulbright Foundation. She persued her dissertation
research in the laboratory of Dr. Ann Progulske and was supported in
part by the Division of Sponsored Research and a training grant from
the National Institutes of Health. Somying plans to continue a
molecular study of the pathogens which cause important infectious
diseases in Thailand.
118

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope arid quality, as dissertation for the degree
of Doctor of Philosophy.
Q£S {¿mSjk 1
Ann Progulske,/fhairman
Assistant Profpdsor of Immunology and
Medical Microbiology
I certify that 1 have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as dissertation for the degree
of Doctor of Philosophy.
/
Ik
Clay B. "walker
Associate Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as dissertation for the degree
of Doctor of Philosophy.
Donna H. Duckworth
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as dissertation for the degree
of Doctor of Philosophy.
William B. Clark
Professor of Immunology and Medical
Microbiology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
William P. McArthur
Professor of Immunology and Medical
Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Anthony F. Barbet
Associate Professor of Veterinary
Medicine
This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
April, 1988
Dean, College of Medicine
Dean, (Graduate School

UNIVERSITY OF FLORIDA
3 1262 08554 4913




PAGE 1

02/(&8/$5 &/21,1* $1' &+$5$&7(5,=$7,21 2) 3$&7(52,'(6 *,1*,9$/,6 $17,*(16 %< 620<,1* 780:$6251 $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

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n JUDGXDWH VWXGHQWV DQG VFLHQWLVWV 'U &RQQLH
PAGE 3

HQFRXUDJHPHQW WKHLU ORYH FRQFHUQ DQG GHYRWLRQ )LQDOO\ ZLVK WR H[WHQG P\ DSSUHFLDWLRQ WR P\ KXVEDQG 6RUQWKHS DQG VRQV 3DWWDUDZXWK DQG 1DWWDSRO IRU WKHLU ORYH SDWLHQFH HQFRXUDJHPHQW DQG GHGLFDWLRQ WKDW FUHDWHG WKH QHFHVVDU\ HQYLURQPHQW WR SHUPLW WKH FRQFOXVLRQ RI WKLV ZRUN

PAGE 4

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

PAGE 5

/,67 2) 7$%/(6 7DEOH 3DJH &KDUDFWHUL]DWLRQ RI ( FROL WUDQVIRUPDQWV ZKLFK H[SUHVV % JLQJLYDO LV DQWLJHQV 7LWHU RI DQWLeE JLQJLYDOLV DJDLQVW ( FROL WUDQVIRUPDQWV ZKLFK H[SUHVV % JLQJLYDOLV DQWLJHQV ,QKLELWLRQ RI DGKHUHQFH WR 6+$ E\ DGVRUEHG DQWLIL JLQJLYDOLV DQWLVHUD ,QKLELWLRQ RI KHUQDJJOXWLQDWLQJ DFWLYLW\ RI % JLQJLYDOLV E\ DQWLKHPDJJOXWLQDWLQJ ( FROL DQWLVHUD ,QKLELWLRQ RI KHUQDJJOXWLQDWLQJ DFWLYLW\ RI % JLQJLYDOLV E\ DGVRUEHG DQWLIE JLQJLYDOLV DQWLVHUXP Y

PAGE 6

/,67 2) ),*85(6 )LJXUH 3DJH 0DS RI S8& $JDURVH JHO HOHFWURSKRUHVLV RI UHFRPELQDQW SODVPLGV $JDURVH JHO HOHFWURSKRUHVLV RI GLIIHUHQW UHVWULFWLRQ GLJHVWV RI UHFRPELQDQW SODVPLG IURP FORQH $JDURVH JHO HOHFWURSKRUHVLV RI GLIIHUHQW UHVWULFWLRQ GLJHVWV RI UHFRPELQDQW SODVPLGV IURP FORQHV DQG $JDURVH JHO HOHFWURSKRUHVLV RI GLIIHUHQW UHVWULFWLRQ GLJHVWV RI UHFRPELQDQW SODVPLGV IURP FORQHV DQG +\EULGL]DWLRQ RI UHFRPELQDQW SODVPLGV ZLWK =3 ODEHOHG % JLQJLYDOLV '1$ SUREH 6'63$*( RQ b DFU\ODPLGHf DQG :HVWHUQ EORW DQDO\VLV RI H[SUHVVHG % JLQJLYDOLV DQWLJHQV 6'63$*( RQ b DFU\ODPLGHf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

PAGE 7

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

PAGE 8

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f GRHV QRW &ORQHV DQG ZnHUH IRXQG WR KDYH RQH LQVHUW IUDJPHQW LQ FRPPRQ DQG WKLV LQVHUW ZDV IRXQG WR KDYH OLWWOH RU QR KRPRORJ\ WR WKH LQVHUW RI FORQH &ORQH LV DOVR DEOH YL L L

PAGE 9

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f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

PAGE 10

&+$37(5 21( ,1752'8&7,21 3HULRGRQWDO GLVHDVH 3'f LV D FKURQLF LQIODPPDWRU\ GLVHDVH ZKLFK UHVXOWV LQ WKH GHVWUXFWLRQ RI WKH VXSSRUWLQJ WLVVXHV RI WHHWK .DJDQ f $OWKRXJK WKH VSHFLILF PLFURELDO HWLRORJ\ RI 3' LV QRW NQRZQ LW LV ZLGHO\ DFFHSWHG WKDW EDFWHULD DUH WKH FRQWULEXWLQJ DJHQWV RI WKH GLVHDVH IRU WKH IROORZLQJ UHDVRQV 6RFUDQVN\ f f GLVHDVH FRUUHODWHV ZLWK WKH SUHVHQFH RI SODTXH f DQWLELRWLFV DUH HIIHFWLYH LQ WUHDWPHQW RI 3' DQG f LPSODQWDWLRQ RI FHUWDLQ JHQHUD RI EDFWHULD LQWR JQRWRELRWLF UDWV UHVXOWV LQ 3' RI LQIHFWHG EXW QRW RI FRQWURO UDWV %DFWHURLGHV JLQJLYDOLV DV WKH 3HULRGRQWRSDWKRJHQ 7KH SUHVHQFH RI D FRPSOH[ PLFURIORUD LQ WKH VXEJLQJLYDO FUHYLFH KDV FRPSOLFDWHG WKH LGHQWLILFDWLRQ RI WKH VSHFLILF HWLRORJLF DJHQWV RI 3,f +RZHYHU VHYHUDO VWXGLHV 6RFUDQVN\ 6ORWV :KLWH DL G 0D\UDQG f LQGLFDWH WKDW D IHZ JHQHUD SULPDULO\ *UDPQHJDWLYH DQDHUREHV DSSHDU WR EH DVVRFLDWHG ZLWK GLVHDVH SURJUHVVLRQ )RU H[DPSOH WKH SURSRUWLRQ RI *UDPQHJDWLYH DQDHUREHV HVSHFLDOO\ EODFN SLJPHQWHG %DFWHURLGHV LQFUHDVHV PDUNHGO\ LQ WKH VXEJLQJLYDO IORUD ZLWK LQFUHDVLQJ VHYHULW\ RI 3' %DFWHURLGHV JLQJLYDOLV SUHYLRXVO\ RUDO %DFWHURLGHV DVDFFKDURO\WLFXV &R\NHQGDOO HW DO f LV WKH EODFN SLJPHQWHG %DFWHURLGHV ZKLFK KDV HPHUJHG DV D NH\ SXWDWLYH SHULRGRQWRSDWKRJHQ IRU D QXPEHU RI FRPSHOOLQJ UHDVRQV

PAGE 11

% JLQJLYDOLV LV WKH SUHGRPLQDQW EDFWHULDO VSHFLHV LVRODWHG IURP SHULRGRQWDO OHVLRQV RI SDWLHQWV ZLWK VHYHUH DGXOW SHULRGRQWLWLV 6ORWV 7DQQHU HW DO f 3DWLHQWV ZLWK DGXOW SHULRGRQWLWLV KDYH EHHQ IRXQG WR KDYH KLJKHU OHYHOV RI ,J* DQWLERGLHV WR % JLQJLYDOLV WKDQ QRUPDO DGXOWV 0RXWRQ HW DO f DQG ORFDO LPPXQLW\ WR % JLQJLYDOLV LV JUHDWHU LQ WKH PRUH DGYDQFHG FDVHV WKDQ LQ WKH HDUO\ IRUPV RI 3' .DJDQ f 6HUXP DQWLERG\ WLWHUV WR % JLQJLYDOLV KDYH EHHQ UHSRUWHG WR GHFUHDVH DIWHU WKHUDS\ RI DGXOW SHULRGRQWLWLV SDWLHQWV VXJJHVWLQJ WKDW DQWLERGLHV WR % JLQJLYDOLV UHVXOW IURP LQIHFWLRQ RI WKLV RUJDQLVP 7ROR HW DO f % JLQJLYDOLV LV DOVR WKH PRVW LQWHUHVWLQJ DQG SRWHQWLDOO\ YLUXOHQW EDFWHULXP FXOWLYDEOH IURP WKH VXEJLQJLYDO FUHYLFH ZLWK UHVSHFW WR LWV FDSDFLW\ IRU EUHDNGRZQ RI WLVVXHV DQG KRVW GHIHQVH PHFKDQLVPV 0D\UDQG DQG 0F%ULGH 9DQ 6WHHQEHUJHQ HW DO 1LOVVRQ HW DO f ,Q DGGLWLRQ % JLQJLYDOLV DSSHDUV WR EH D FDXVDWLYH DJHQW RI H[SHULPHQWDO SHULRGRQWLWLV LQ DQLPDOV :KHQ % JLQJLYDOLV LV LPSODQWHG DV WKH PRQRFRQWDPLQDQW LQ JQRWRELRWLF UDWV LW FDXVHV DFFHOHUDWHG DOYHRODU ERQH ORVV &UDZ7RUG HW DO f ,Q D ORQJLWXGLQDO VWXG\ RI DOYHRODU ERQH ORVV LQ 0DFDFD DUFWRLGHV 6ORWV DQG +DXVPDQQ f WKH SURSRUWLRQ RI % JLQJLYDOLVW\SH LVRODWHV UHSRUWHGO\ LQFUHDVHG IURP D PLQRULW\ RI WKH FXOWLYDEOH PLFURELRWD SULRU WR ERQH ORVV WR D PDMRULW\ RI WKH PLFURIORUD ZKHQ DOYHRODU ERQH ORVV ZDV GHWHFWDEOH 3DWKRJHQLFLW\ RI % JLQJLYDOLV $OWKRXJK % JLQJLYDOLV KDV EHHQ VWURQJO\ LPSOLFDWHG DV DQ HWLRORJLFDO DJHQW RI DGXOW SHULRGRQWLWLV LWV H[DFW UROH LQ WKH GLVHDVH SURFHVV KDV QRW \HW EHHQ HVWDEOLVKHG ,Q RUGHU WR SURGXFH 3' LW LV

PAGE 12

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f &RORQL]DWLRQ ,W LV QRZ UHFRJQL]HG WKDW FRORQL]DWLRQ RI WKH RUDO FDYLW\ DQG PDQ\ RWKHU PXFRVDO HQYLURQPHQWV UHTXLUHV WKH DGKHUHQFH RI EDFWHULD WR WKH VXUIDFH LQ RUGHU WR UHVLVW WKH FOHDQVLQJ DFWLRQ RI JODQGXODU VHFUHWLRQV *LEERQV DQG 9DQ +RXWH f 7KH DGKHUHQFH RI EDFWHULD WR KRVW WLVVXHV LV WKXV D SUHUHTXLVLWH IRU FRORQL]DWLRQ ZKLFK LV WKH LQLWLDO HYHQW LQ WKH SDWKRJHQHVLV RI GLVHDVH *LEERQV DQG 9DQ +RXWH f 7KH PHFKDQLVPV RI EDFWHULDO DGKHUHQFH LQYROYH ERWK LRQLF DQG RWKHU SK\VLFDO FRYDOHQWf IRUFHV 0DQ\ LI QRW DOO SDWKRJHQLF EDFWHULD SRVVHVV VSHFLILF OLJDQGV RQ WKHLU VXUIDFHV FDOOHG DGKHVLQV ZKLFK ELQG WR FRPSOHPHQWDU\ FRPSRQHQWV RQ KRVW WLVVXHV *LEERQV DQG 9DQ +RXWH f 7KH PHFKDQLVPV RI DGKHUHQFH PD\ LQYROYH WKH LQWHUDFWLRQ RI FDUERK\GUDWH ELQGLQJ SURWHLQV RU OHFWLQV RQ EDFWHULDO VXUIDFHV ZLWK FDUERK\GUDWHFRQWDLQLQJ UHFHSWRUV RQ KRVW FHOOV %LQGLQJ SURSHUWLHV RI DGKHVLQV PD\ DOVR EH IDFLOLWDWHG E\ WKHLU K\GURSKRELF GRPDLQV *LEERQV f

PAGE 13

&RPSRQHQWV RI EDFWHULD ZKLFK PHGLDWH DWWDFKPHQW WR KRVW WLVVXHV LQFOXGH VXUIDFH VWUXFWXUHV VXFK DV ILPEULDH FDSVXODU PDWHULDOV OLSRSRO\VDFFKDULGHV DQG PHPEUDQHDVVRFLDWHG H[WUDFHOOXODU YHVLFOHV 6ORWV DQG *HQFR f ,Q WKH RUDO FDYLW\ EDFWHULD FDQ DWWDFK WR KRVW WLVVXHV DV ZHOO DV *UDPSRVLWLYH EDFWHULD LQ SUHIRUPHG SODTXH 6ORWV DQG *LEERQV f 7KH QDWXUH RI WKH ELQGLQJ VLWHV RQ WHHWK DQG RUDO WLVVXHV WR ZKLFK *UDPQHJDWLYH EDFWHULD DWWDFK KDV QRW EHHQ ZHOO HVWDEOLVKHG ,Q YLWUR % JLQJLYDOLV FDQ DWWDFK WR DQG DJJOXWLQDWH HU\WKURF\WHV 2NXGD DQG 7DND]RH 6ORWV DQG *LEERQV U 6ORWV DQG *HQFR 2NXGD HW DO f FDQ DGKHUH LQ KLJK QXPEHUV WR KXPDQ EXFFDO HSLWKHOLDO FHOOV 6ORWV DQG *LEERQV 2NXGD HW DO f FUHYLFXODU HSLWKHOLDO FHOOV GHULYHG IURP SHULRGRQWDO SRFNHWV 6ORWV DQG *LEERQV f DQG VXUIDFHV RI *UDP SRVLWLYH EDFWHULD SUHVHQW LQ SODTXH 6ORWV DQG *LEERQV 6FKZDU] HW DO f % JLQJLYDOLV LV DOVR DEOH WR DGKHUH WR XQWUHDWHG DQG VDOLYDWUHDWHG K\GUR[\DSDWLWH EXW LQ FRPSDUDWLYHO\ ORZ QXPEHUV 6ORWV DQG *LEERQV f % JLQJLYDOLV KDV DOVR EHHQ UHSRUWHG WR ELQG WR +5 PDWUL[ D PDWHULDO VLPLODU WR WKH EDVHPHQW PHPEUDQH EDUULHU XQGHUO\LQJ FRQQHFWLYH WLVVXH /HRQJ HW DO f 5HFHQWO\ LW KDV EHHQ UHSRUWHG WKDW % JLQJLYDOLV FDQ ELQG ILEULQRJHQ DQG SRVVLEO\ FRORQL]H KRVW WLVVXHV E\ DWWDFKLQJ WR ILEULQRJHQFRDWHG VXUIDFHV /DQW] HW DO f %DFWHULDO DQWDJRQLVP PD\ DOVR SOD\ DQ LPSRUWDQW UROH LQ PHGLDWLQJ WKH FRORQL]DWLRQ RI % JLQJLYDOLV ,Q QRUPDO DGXOWV 6WUHSWRFRFFXV VDQJXLV LV D SUHGRPLQDQW RUJDQLVP LQ VXSUD DQG VXEJLQJLYDO SODTXH 6 VDQJXLV HODERUDWHV VDQJXLFLQ D EDFWHULRFLQ ZKLFK LQ YLWUR LQKLELWV EODFNSLJPHQWHG %DFWHULRGHV 1DNDPXUD HW DO f ([SHULPHQWDO VWXGLHV LQ KXPDQV KDYH VKRZQ WKDW WKH QXPEHU RI 6WUHSWRFRFFXV VSHFLHV

PAGE 14

LQFOXGLQJ VDQJXLV DUH GHFUHDVHG ZKLOH WKRVH RI $FWLQRP\FHV DQG EODFN SLJPHQWHG %DFWHULRGHV DUH LQFUHDVHG /HRVFKH DQG 6\HG f LQ JLQJLYLWLV 7KH PHFKDQLVP RI WKH SURSRUWLRQDO GHFUHDVH RI 6 VDQJXLV LV QRW NQRZQ EXW WKLV VKLIW VHHPV WR EH RQH RI WKH WULJJHUV IRU WKH LQLWLDWLRQ RI FRPSRVLWLRQDO FKDQJHV LQ WKH VXEJLQJLYDO IORUD $ GHFUHDVH RI VDQJXLFLQ SURGXFWLRQ PD\ SHUPLW WKH JURZWK RI $FWLQRP\FHV VSHFLHV DQG EODFN SLJPHQWHG %DFWHULRGHV 7DND]RH HW DO f 7KH JURZWK RI % JLQJLYDLLV PD\ EH HQKDQFHG E\ KHUULLUL ZKHQ EOHHGLQJ RFFXUV LQ JLQJLYLWLV VLQFH KHPLUL LV D UHTXLUHG IDFWRU IRU WKH FXOWLYDWLRQ RI % JLQJLYDLLV 5HFHQWO\ LW KDV EHHQ UHSRUWHG WKDW % JLQJLYDLLV JURZQ XQGHU KHPLQOLPLWHG FRQGLWLRQV KDV D UHGXFHG YLUXOHQFH LQ PLFH FRPSDUHG ZLWK EDFWHULD FXOWXUHG LQ DQ H[FHVV RI KHPLQ 0F.HH HW DO f :KHQ FRORQL]DWLRQ RI % JLQJLYDLLV RFFXUV WKHUH VHHPV WR EH D FKDQJH LQ WKH EDFWHULDO FRPSRVLWLRQ LQ WKH SHULRGRQWDO SRFNHW 7KLV FRXOG EH H[SODLQHG E\ VWXGLHV RI 1DNDPXUD HW DO f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f

PAGE 15

(YDVLRQ RI +RVW 'HIHQVH % JLQJLYDOLV PD\ VXUYLYH LQ WKH SHULRGRQWDO SRFNHW EHFDXVH LW UHVLVWV SKDJRF\WRVLV 6XQGTYLVW HW DO f GHPRQVWUDWHG WKDW LQ YLWUR PRVW VWUDLQV RI % JLQJLYDOLV H[KLELW D KLJKHU UHVLVWDQFH WR SKDJRF\WRVLV WKDQ GR OHVV SDWKRJHQLF VWUDLQV DQG WKDW LPSDLUHG SKDJRF\WRVLV RI WKLV EDFWHULDO VSHFLHV LV UHODWHG WR FDSVXODU PDWHULDO 7KH %DFWHURLGHV FDSVXOH RQO\ SRRUO\ DFWLYDWHV FRPSOHPHQW WKHUHIRUH LW PD\ IXQFWLRQ WR GHFUHDVH 301 FKHPRWDFWLF VWLPXOXV E\ PDVNLQJ /36 ZKLFK VWURQJO\ DFWLYDWHV FRPSOHPHQW 2NXGD HW DO f 9DULRXV H[SHULPHQWV KDYH YHULILHG WKDW WKH EODFN SLJPHQWHG %DFWHURLGHV VWUDLQV GR QRW VWLPXODWH D VWURQJ 301 FKHPRWDFWLF UHVSRQVH 6YHHQ DE /LQGKH DQG 6RFUDQVN\ 6XQGTYLVW DQG -RKDQVVRQ f 0RVW VWUDLQV RI % JLQJLYDOLV GHPRQVWUDWH UHVLVWDQFH WR VHUXP EDFWHULFLGDO V\VWHPV 6XQGTYLVW DQG -RKDQVVRQ f % JLQJLYDOLV KDV DOVR EHHQ VKRZQ WR GHJUDGH WKH SODVPD SURWHLQV ZKLFK DUH LPSRUWDQW LQ WKH KRVW GHIHQVH VXFK DV WKH FRPSOHPHQW IDFWRUV & DQG & 6XQGTYLVW HW DO f LPPXQRJOREXOLQV $ DQG 0 .LOLDQ 6XQGTYLVW HW DO f DOSKDOSURWHLQDVH LQKLELWRU DOSKDPDFURJOREXLLQ &DUOVVRQ HW DO Df KDSWRJORELQ DQG KHPRSH[LQ &DUOVVRQ HW DO Ef ,W KDV DOVR EHHQ VKRZQ WKDW % JLQJLYDOLV KDV WKH FDSDFLW\ WR LQDFWLYDWH DQG GHJUDGH WKH SODVPD SURWHLQV RI LPSRUWDQFH LQ WKH LQLWLDWLRQ DQG FRQWURO RI WKH LQIODPPDWRU\ UHVSRQVH VXFK DV &O LQKLELWRU DQWLWKURPELQ DQG DLSKD DQWLSODVPLQ 1LOVVRQ HW DO f ,Q DGGLWLRQ % JLQJLYDOLV FDQ GHJUDGH ILEULQRJHQ /DQW] HW DO f DQG ILEULQ 0D\UDQG DQG 0F%ULGH :LNVWURP HW DO f WKHUHIRUH QR HIIHFWLYH ILEULQ EDUULHU LV IRUPHG DURXQG WKH RUJDQLVP % JLQJLYDOLV WKXV DSSHDUV WR

PAGE 16

EH DQ RUJDQLVP IXOO\ FDSDEOH RI LQDFWLYDWLQJ WKH KRVW GHIHQVH PHFKDQLVPV DJDLQVW LQYDGLQJ EDFWHULD 3HULRGRQWDO 7LVVXH 'HVWUXFWLRQ % JLQJLYDOLV SRVVHVVHV D QXPEHU RI FRPSRQHQWV ZLWK WKH SRWHQWLDO WR GHVWUR\ JLQJLYDO WLVVXH FRQVWLWXHQWV DV IROORZV 7KH % JLQJLYDOLV OLSRSRO\VDFFKDULGH SRVVHVVHV VWURQJ ERQH UHVRUSWLYH DFWLYLW\ 1DLU HW DO f DQG LQKLELWV WKH JURZWK RI FXOWXUHG ILEUREODVWV GHULYHG IURP KHDOWK\ DQG SHULRGRQWDOO\ GLVHDVHG KXPDQ JLQJLYD /D\PDQ DQG 'LHGULFK f 7KH OLSRSRO\VDFFKDULGH LV DOVR D VXVSHFWHG FRPSRQHQW WKDW VWLPXODWHV PRQRQXFOHDU FHOOV WR SURGXFH D IDFWRU ZKLFK VWURQJO\ VWLPXODWHV RVWHRFODVWPHGLDWHG PLQHUDO UHVRUSWLRQ %RP9DQ 1RRUORRV HW DO f % JLQJLYDOLV SURWHRO\WLF HQ]\PHV HVSHFLDOO\ FROODJHQDVH 0D\UDQG DQG 0F%ULGH 5REHUWVRQ HW DO 0D\UDQG DQG *UHQLHU f DQG D WU\SVLQOLNH SURWHDVH 6ORWV /DXJKRQ HW DO f PD\ EH GLUHFWO\ LQYROYHG LQ SHULRGRQWDO WLVVXH GHVWUXFWLRQ (Q]\PHV RWKHU WKDQ SURWHDVHV PD\ DOVR SOD\ DQ LPSRUWDQW UROH LQ WKH SDWKRJHQHVLV RI SHULRGRQWDO GLVHDVH )RU H[DPSOH DONDOLQH DQG DFLG SKRVSKDWDVHV 6ORWV /DXJKRQ HW DO f PD\ FDXVH DOYHRODU ERQH EUHDNGRZQ VLQFH LW KDV EHHQ VKRZQ WKDW EDFWHULDO SKRVSKDWDVHV FRXOG FDXVH DOYHRODU ERQH EUHDNGRZQ )UDQN DQG 9RHJHO f %DFWHULDO SURGXFWV LH EXW\UDWH SURSLRQDWH 6LQJHU DQG %XFNQHU f DQG YRODWLOH VXOIXU FRPSRXQGV 7RQ]HWLFK DQG 0F%ULGH f DUH DOVR VXVSHFWHG WR EH WR[LF WR SHULRGRQWDO WLVVXHV 5HFHQWO\ LW KDV EHHQ UHSRUWHG WKDW % JLQJLYDOLV SRVVHVVHV D FDUWLODJHGHJUDGLQJ DELOLW\ ZKLFK LV VXVSHFWHG WR EH GXH WR LWV DELOLW\ WR GHJUDGH SURWHLQDVH LQKLELWRUV .ODPIHOGW f

PAGE 17

,W KDV EHHQ VXJJHVWHG WKDW SHULRGRQWDO WLVVXH GHVWUXFWLRQ LV PHGLDWHG QRW RQO\ E\ EDFWHULD DQG WKHLU SURGXFWV EXW DLVR E\ WKH KRVW GHIHQVH PHFKDQLVPV +RUWRQ HW DK 1LVHQJDUG f )RU H[DPSOH FHOO PHGLDWHG LPPXQLW\ KDV EHHQ VKRZQ WR FRUUHODWH ZLWK WKH SHULRGRQWDO VWDWXV RI SDWLHQWV ,YDQ\L DQG /HKQHU 3DWWHUV HW DO 3DWWHUV HW DO f % JLQJLYDOLV ZDV IRXQG WR VWLPXODWH VLJQLILFDQWO\ PRUH O\PSKRSUROLIHUDWLYH UHVSRQVH LUL SDWLHQWV ZLWK GHVWUXFWLYH SHULRGRQWLWLV WKDQ WKRVH RI QRUPDO VXEMHFWV RU WKRVH ZLWK JLQJLYLWLV 3DWWHUV HW DO f $ O\PSKRSUROLIHUDWLYH UHVSRQVH UHVXOWV LQ WKH SURGXFWLRQ RI O\PSKRNLQHV VHYHUDO RI ZKLFK FDQ DFFRXQW IRU VRPH RI WKH GHVWUXFWLYH HIIHFWV LQ SHULRGRQWDO GLVHDVH )RU H[DPSOH DOSKDO\PSKRWR[LQ FDQ FDXVH FHOO GHDWK DQG RVWHRFODVW DFWLYDWLQJ IDFWRU FDQ VWLPXODWH RVWHRFODVWLF ERQH UHVRUSWLRQ +RUWRQ HW DO f 0DFURSKDJHV KDYH DOVR EHHQ VXJJHVWHG WR SOD\ D UROH LQ SHULRGRQWDO WLVVXH GHVWUXFWLRQ 0DFURSKDJHV PD\ EH VWLPXODWHG E\ EDFWHULDO DQWLJHQV VXFK DV /36 :DKO f RU E\ O\PSKRNLQHV 0RRQH\ DQG :DNVPDQ :DKO HW DO f DQG VXEVHTXHQWO\ SURGXFH WLVVXHGHJUDGLQJ HQ]\PHV VXFK DV FROODJHQDVH DQG RWKHU SURWHDVHV :DKO HW DO :DKO f 5HFHQWO\ LW KDV EHHQ GHPRQVWUDWHG WKDW OLSRSRO\VDFFKDULGH RI % JLQJLYDOLV FDQ LQGXFH FLUFXODWLQJ PRQRQXFOHDU FHOOV WR UHOHDVH FROODJHQDVHLQGXFLQJ F\WRNLQHV 7KH F\WRNLQHV WKHQ LQGXFH FROODJHQDVH V\QWKHVLV LQ KXPDQ JLQJLYDO ILEUREODVWV +HDOWK HW DO f ,Q DGGLWLRQ ,J( PDVW FHOOV DQG EDVRSKLOV PD\ DOVR SOD\ D UROH LQ SHULRGRQWDO GLVHDVH -D\DZDUGHQH DQG *ROGQHU 2OVVRQ :HQQVWURP HW DO f

PAGE 18

$SSOLFDWLRQ RI 5HFRPELQDQW '1$ 7HFKQLTXHV WR WKH 6WXG\ RI 3HULRGRQWDO 'LVHDVH 7KH UHFRPELQDQW '1$ WHFKQLTXHV GHYHORSHG GXULQJ WKH SDVW IHZ \HDUV KDYH SURYHQ W EH SRZHUIXO WRROV IRU WKH VWXG\ RI SDWKRJHQHVLV 6HYHUDO PDMRU DQWLJHQV DQG YLUXOHQFH IDFWRUV KDYH EHHQ FORQHG DV D PHDQV RI IXUWKHU FKDUDFWHUL]LQJ WKHLU FKHPLFDO QDWXUHV JHQHWLF UHJXODWLRQ DQG IXQFWLRQ LQ YDULRXV GLVHDVHV )RU H[DPSOH WKH FORQLQJ DQG H[SUHVVLRQ RI WKH 1HLVVHULD JRQRUUKRHDH SLOXV SURWHLQ LQ ( FROL 0H\HU DQG 6R f KDV KHOSHG H[SODLQ WKH PROHFXODU PHFKDQLVP RI DQWLJHQLF YDULDWLRQ ,Q RWKHU VWXGLHV WKH FORQLQJ RI VHYHUDO YLUXOHQFH IDFWRUV LQFOXGLQJ H[RWR[LQV 9RGNLQ DQG /HSSOD 9DVLO HW DO 1LFRVLD HW DO f HQWHURWR[LULV 3HDUVRQ DQG 0HNDODQRV f D KHPRO\VLQ *ROGEHUJ DQG 0XUSK\ f DQG D SQHXPRO\VLQ :DONHU HW DO f KDYH DOORZHG JHQHWLF VWXGLHV RI WKHVH SURWHLQV DQG KDYH IDFLOLWDWHG WKH SURGXFWLRQ RI VDIHU YDFFLQHV &ORQLQJ DQWLJHQV HQFRGHG E\ XQNQRZQ JHQHV LV PDGH SRVVLEOH E\ SUHSDULQJ D JHQRPLF OLEUDU\ LQ ZKLFK DQ\ JHQH LV WKHRUHWLFDOO\ UHSUHVHQWHG ,I WKH QXPEHU RI FORQHV LV ODUJH HQRXJK LW LV KRSHG WKDW DQ\ JHQH FDQ EH LVRODWHG E\ VFUHHQLQJ WKH OLEUDU\ 3HUEDO f *HQRPLF OLEUDULHV RI ERWK 7UHSRQHPD SDOOLGXP 6WDPP HW DO f DQG /HJLRQHOOD SQHXPRSKLOD (QJOHEHUJ HW DO DEf KDYH EHHQ PDGH DV D ILUVW VWHS LQ LVRODWLQJ DQG FKDUDFWHUL]LQJ WKHLU PDMRU VXUIDFH DQWLJHQV 7KH UHFRPELQDQW '1$ WHFKQLTXHV KDYH KRZHYHU EHHQ DSSOLHG RQO\ VSDULQJO\ WR WKH VWXG\ RI *UDPQHJDWLYH DQDHURELF SDWKRJHQV DQG HYHQ OHVV WR WKH VWXG\ RI WKH PROHFXODU PHFKDQLVPV RI SHULRGRQWRSDWKRJHQHVLV 7KH UHFRPELQDQW '1$ PHWKRGRORJLHV RIIHU DGYDQWDJHV RYHU SUHYLRXV PHWKRGV XVHG LQ WKH VWXG\ RI RUDO SDWKRJHQV 6LQFH VHYHUDO SRWHQWLDO

PAGE 19

SHULRGRQWRSDWKRJHQV LQFOXGLQJ % JLQJLYD-LV DUH GLIILFXOW WR JURZ WR KLJK GHQVLWLHV LVRODWLRQ DQG SXULILFDWLRQ RI DQWLJHQV HVSHFLDOO\ WKRVH SUHVHQW LQ VPDOO DPRXQWV DUH RIWHQ GLIILFXOW DQG WHGLRXV EHFDXVH RI D OLPLWHG DPRXQW RI VWDUWLQJ PDWHULDO &ORQLQJ VSHFLILF VWUXFWXUHV LQ DQ RUJDQLVP VXFK DV ( FROL ZRXOG JUHDWO\ DOOHYLDWH WKHVH SUREOHPV VLQFH ( FROL FDQ EH JURZQ WR KLJK GHQVLWLHV HDVLO\ DQG FORQHG VWUXFWXUHV FDQ EH RYHUSURGXFHG LQ ( FROL 'H )UDQFR HW DO 0DWVXPXUD HW DO f 7KLV ZRXOG IDFLOLWDWH WKH LVRODWLRQ DQG SXULILFDWLRQ RI WKDW VWUXFWXUH RU FRPSRQHQW $OVR WKH FORQLQJ DQG H[SUHVVLRQ RI DQWLJHQV ZRXOG LVRODWH WKH DQWLJHQV DW WKH JHQHWLF OHYHO 7KH FORQHG DQWLJHQV FDQ WKHQ EH SUHSDUHG DV SURGXFWV GHYRLG RI RWKHU % JLQJLYDOLV DQWLJHQV 7KLUGO\ WKH FORQLQJ RI % JLQJLYDOLV DQWLJHQV ZRXOG DOORZ D JHQHWLF DQG PROHFXODU DQDO\VLV RI WKH JHQHVf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f RI % JLQJLYDOLV FKURPRVRPDO '1$ LQ ( FROL ,GHQWLI\ ( FROL WUDQVIRUPDQWV ZKLFK H[SUHVV % JLQJLYDOLV DQWLJHQV

PAGE 20

,GHQWLI\ FORQHG DQWLJHQV ZKLFK DUH SRWHQWLDO YLUXOHQFH IDFWRUV

PAGE 21

&+$37(5 7:2 &/21,1* $1' (;35(66,21 2) %$&7(52:(6 *,1*,9$/,6 $17,*(16 ,1 (6&+(5,&+,$ &2/, ,QWURGXFWLRQ 6HYHUDO OLQHV RI HYLGHQFH VWURQJO\ LPSOLFDWH %DFWHURLGHV JLQJLYDOLV D *UDPQHJDWLYH DQDHURELF EDFWHULXP DV DQ HWLRORJLFDO DJHQW RI DGXOW SHULRGRQWDO GLVHDVH :KLWH DQG 0D\UDULG =DPEQ HW DO 7DND]RH HW DO 6ORWV DQG *HQFR 6ORWV HW DO f )RU H[DPSOH UHODWLYHO\ KLJK SURSRUWLRQV RI % JLQJLYDOLV KDYH EHHQ LVRODWHG IURP DGXOW SHULRGRQWLWLV OHVLRQV 6ORWV 7DQQHU HW DO 6SLHJHO HW DO f SDWLHQWV ZLWK DGXOW SHULRGRQWLWLV KDYH EHHQ IRXQG WR KDYH KLJKHU OHYHOV RI ,J* DQWLERGLHV WR % JLQJLYDOLV WKDQ GR QRUPDO DGXOWV 0RXWRQ HW DO 1DLWR HW DO f DQG ORFDO LPPXQLW\ WR % JLQJLYDOLV LV JUHDWHU LQ WKH PRUH DGYDQFHG FDVHV WKDQ LQ WKH HDUO\ IRUPV RI SHULRGRQWDO GLVHDVH .DJDQ f % JLQJLYDOLV DOVR DSSHDUV WR EH D FDXVDWLYH DJHQW RI H[SHULPHQWDO SHULRGRQWLWLV LQ DQLPDOV &UDZIRUG HW DO 6ORWV DQG +DXVPDQQ f ,Q DGGLWLRQ % JLQJLYDOLV SRVVHVVHV D YDULHW\ RI VXVSHFWHG YLUXOHQFH IDFWRUV VXFK DV SURWHDVHV FROODJHQDVHV LPPXQRORJOREXLLQ GHJUDGLQJ HQ]\PHV DQG DGKHVLQV 6ORWV DQG *HQFR f 3UHYLRXV LQYHVWLJDWLRQV RI %DFWHURLGHV SDWKRJHQLF PHFKDQLVPV KDYH HPSOR\HG WKH LVRODWLRQ DQG SXULILFDWLRQ RI % JLQJLYDOLV FRQVWLWXHQWV E\

PAGE 22

% JLQJLYDOLV LV WKH SUHGRPLQDQW EDFWHULDO VSHFLHV LVRODWHG IURP SHULRGRQWDO OHVLRQV RI SDWLHQWV ZLWK VHYHUH DGXOW SHULRGRQWLWLV 6ORWV 7DQQHU HW DO f 3DWLHQWV ZLWK DGXOW SHULRGRQWLWLV KDYH EHHQ IRXQG WR KDYH KLJKHU OHYHOV RI ,J* DQWLERGLHV WR % JLQJLYDMLV WKDQ QRUPDO DGXOWV 0RXWRQ HW DO f DQG ORFDO LPPXQLW\ WR % JLQJLYDOLV LV JUHDWHU LQ WKH PRUH DGYDQFHG FDVHV WKDQ LQ WKH HDUO\ IRUPV RI 3' .DJDQ f 6HUXP DQWLERG\ WLWHUV WR % JLQJLYDOLV KDYH EHHQ UHSRUWHG WR GHFUHDVH DIWHU WKHUDS\ RI DGXOW SHULRGRQWLWLV SDWLHQWV VXJJHVWLQJ WKDW DQWLERGLHV WR % JLQJLYDOLV UHVXOW IURP LQIHFWLRQ RI WKLV RUJDQLVP 7ROR HW DO f % JLQJLYDOLV LV DOVR WKH PRVW LQWHUHVWLQJ DQG SRWHQWLDOO\ YLUXOHQW EDFWHULXP FXOWLYDEOH IURP WKH VXEJLQJLYDO FUHYLFH ZLWK UHVSHFW WR LWV FDSDFLW\ IRU EUHDNGRZQ RI WLVVXHV DQG KRVW GHIHQVH PHFKDQLVPV 0D\UDQG DQG 0F%ULGH 9DQ 6WHHQEHUJHQ HW DO 1LOVVRQ HW DO f ,Q DGGLWLRQ % JLQJLYDOLV DSSHDUV WR EH D FDXVDWLYH DJHQW RI H[SHULPHQWDO SHULRGRQWLWLV LQ DQLPDOV :KHQ % JLQJLYDOLV LV LPSODQWHG DV WKH PRQRFRQWDPLQDQW LQ JQRWRELRWLF UDWV LW FDXVHV DFFHOHUDWHG DOYHRODU ERQH ORVV &UDXnIRUG HW DO f ,Q D ORQJLWXGLQDO VWXG\ RI DOYHRODU ERQH ORVV LQ 0DFDFD DUFWRLGHV 6ORWV DQG +DXVPDQQ f WKH SURSRUWLRQ RI % JLQJLYDOLVW\SH LVRODWHV UHSRUWHGO\ LQFUHDVHG IURP D PLQRULW\ RI WKH FXOWLYDEOH PLFURELRWD SULRU WR ERQH ORVV WR D PDMRULW\ RI WKH PLFURIORUD ZKHQ DOYHRODU ERQH ORVV ZDV GHWHFWDEOH 3DWKRJHQLFLW\ RI % JLQJLYDOLV $OWKRXJK % JLQJLYDOLV KDV EHHQ VWURQJO\ LPSOLFDWHG DV DQ HWLRORJLFDO DJHQW RI DGXOW SHULRGRQWLWLV LWV H[DFW UROH LQ WKH GLVHDVH SURFHVV KDV QRW \HW EHHQ HVWDEOLVKHG ,Q RUGHU WR SURGXFH )' LW LV

PAGE 23

0DWHULDOV DQG 0HWKRGV %DFWHULD@ 6WUDLQV 3ODVU G DQG *URZWK &RQGLWLRQV %DFWHURLGHV JLQJLYD-LV REWDLQHG IURP D VWRFN FXOWXUH ZDV JURZQ RQ SODWHV FRQWDLQLQJ 7U\SWLFDVH VR\ DJDU %%/ 0LFURELRORJ\ 6\VWHPV &RFNH\VYLOOH 0Gf VXSSOHPHQWHG ZLWK VKHHS EORRG bf KHPLQ PLFURJUDPV SHU POf DQG PHQDGLRQH PLFURJUDPV SHU POf 7KH RUJDQLVP ZDV DOVR JURZQ LQ PO RI 7RGG+HZLWW EURWK %%/f VXSSOHPHQWHG ZLWK KHPLQ PLFURJUDPV SHU POf PHQDGLRQH PLFURJUDPV SHU POf DQG JOXFRVH PLOOLJUDPV SHU POf &XOWXUHV ZHUH LQFXEDWHG LQ DQ DQDHURELF 2 FKDPEHU LQ D 1+&2 f DWPRVSKHUH DW & XQWLO WKH ORJ SKDVH RI JURXnWK ZDV REWDLQHG 7KH PO EURWK FXOWXUH ZDV WUDQVIHUUHG LQWR PO RI WKH VDPH PHGLXP DQG VXEVHTXHQWO\ WUDQVIHUUHG WR PO RI 2 PHGLXP ,QFXEDWLRQ ZDV DW & DQDHURELFDOO\ XQWLO D ODWH ORJ SKDVH FXOWXUH ZnDV REWDLQHG ( FROL -0 UHF $O HQG $O J\U $ WKL KVG 5 VXS ( UHO$O $ODFSUR $%f &)WUD SUR$% ODF ,= 0@f DQG WKH SODVPLG H[SUHVVLRQ YHFWRU S8& )LJXUH f ZHUH JLIWV RI 0HVVLQJ DQG KDYH EHHQ GHVFULEHG SUHYLRXVO\ 9LHLUD DQG 0HVVLQJ
PAGE 24

)LJXUH 0DS RI S8&

PAGE 25

+LQG ,,, 3VW 6DO %DP +, 6PD (FR 5O S8& EDVH SDLUVf

PAGE 26

3UHSDUDWLRQ R &KURPRVRPDO '1$ IURP % JLLLJLYD+V &KURPRVRPDO '0$ IURP 3 JLQJLYDOLV ZDV SUHSDUHG E\ WKH PHWKRG RI $ 'DV SHUVRQDO FRPPXQLFDWLRQf DV IROORZV RQH WR WKUHH OLWHUV RI FHOOV ZHUH SHOOHWHG E\ FHQWULIXJDWLRQ DQG ZDVKHG RQFH ZLWK O[ 66& EXIIHU b 1D&nO b 1D FLWUDWHf FRQWDLQLQJ b VXFURVH DQG P0 ('7$ 7KH FHOOV ZHUH SHOOHWHG DQG UHVXVSHQGHG LQ RI WKH RULJLQDO YROXPH RI WKH VDPH EXIIHU DW r& /\VR]\PH PJPOf LQ 66& ZDV DGGHG R WR PJPO WKH PL[WXUH ZDV PL[HG WKRURXJKO\ DQG LQFXEDWHG DW & IRU PLQXWHV 1LQH YROXPHV RI O[ 66& FRQWDLQLQJ b VXFURVH P0 ('7$ DQG b 6'6 SUHXDUUQHG WR &f ZHUH DGGHG DQG WKH FHOO 2 VXVSHQVLRQ ZDV LQFXEDWHG DW & IRU WR PLQXWHV XQWLO FHOO O\VLV ZDV FRPSOHWH ,Q RUGHU WR GHQDWXUH DQ\ FRQWDPLQDWLQJ SURWHLQV SURWHLQDVH ZDV DGGHG WR D ILQDO FRQFHQWUDWLRQ RI PJPO DQG WKH O\VDWH XnDV LQFXEDWHG DW r & IRU KRXUV '1$ ZDV H[WUDFWHG WZLFH ZLWK SKHQRO WZLFH ZLWK SKHQROFKORURIRUP E\ YROXPHf DQG IRXU WLPHV ZLWK FKORURIRUP 7XAR YROXPHV RI DEVROXWH DOFRKRO XnHUH DGGHG DQG WKH SUHFLSLWDWHG '1$ ZDV VSRROHG RQWR D JODVV URG 7KH SXULILHG '1$ ZDV ULQVHG ZLWK b HWKDQRO DQG VXVSHQGHG LQ 7( EXIIHU S+ P0 7ULV+&O S+ P0 ('7$f ,VRODWLRQ RI 3ODVPLG '1$ 3ODVPLG '1$ ZDV LVRODWHG E\ WKH PHWKRG RI ,VK+RURZLF] DQG %XUNH f LQ ZKLFK FHOOV ZUHUH O\VHG ZLWK 6'6('7$ LQ WKH SUHVHQFH RI 1D2+ 3RWDVVLXP DFHWDWH S+ ZDV DGGHG DW r& DQG FHOO GHEULV SURWHLQ 51$ DQG FKURPRVRPDO '1$ Z"HUH UHPRYHG E\ FHQWULIXJDWLRQ 7KH SODVPLG ZUDV SUHFLSLWDWHG ZLWK YROXPHV RI HWKDQRO ZDVKHG ZLWK b HWKDQRO GULHG DQG UHVXVSHQGHG LQ 7( EXIIHU DW S+ 7KH SODVPLG ZDV

PAGE 27

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f *HQRPLF IUDJPHQWV ZHUH DOVR REWDLQHG E\ SDUWLDO GLJHVWLRQ RI WKH FKURPRVRPDO '1$ ZLWK +LQG ,,, UHVWULFWLRQ HQGRQXFOHDVH DQG OLJDWHG WR WKH GHSKRVSKRU\ODWHG +LQG ,,, VLWH RI S8& 7KH UHFRPELQDQW SODVPLGV ZHUH XVHG WR WUDQVIRUP ( FROL -0 E\ WKH PHWKRG RI $ 'DV SHUVRQDO FRPPXQLFDWLRQf %ULHIO\ ( FROL -0 ZDV JURZQ WR DQ HDUO\ ORJ SKDVH 2' f LQ /% EURWK 7HQ PO RI WKH FXOWXUH ZHUH 2 FHQWULIXJHG DW USP IRU PLQXWHV DW & DQG UHVXVSHQGHG LQ PO RI WUDQVIRUPDWLRQ EXIIHU 7)0 P0 7ULV+&O S+ 0 1D&Of 7KH FHOOV ZHUH WKHQ SHOOHWHG DQG UHVXVSHQGHG LQ PO RI 7)0 P0 &D&8f DQG LQFXEDWHG RQ LFH IRU PLQXWHV 7KH FHOOV ZHUH DJDLQ SHOOHWHG DQG JHQWO\ UHVXVSHQGHG LQ PO RI 7)0 DQG GLVSHQVHG LQWR PO DOLTXRWV 2QH WHQWK PO RI 7)0 P0 7ULV+&O S+ P0 &D&O P0 0J62Lf ZDV DGGHG WR HDFK DOLTXRW IROORZHG E\ YDU\LQJ DPRXQWV RI '1$ 7KH FHOOV ZHUH WKHQ DOORZHG WR LQFXEDWH RQ LFH 2 IRU PLQXWHV DQG KHDW VKRFNHG DW & IRU PLQXWHV /% EURWK

PAGE 28

POf ZDV DGGHG DQG WKH FHOO VXVSHQVLRQ ZDV LQFXEDWHG DW r & IRU KRXU )LQDOO\ WKH FHOOV ZHUH SODWHG RQ /% DJDU FRQWDLQLQJ DPSLFLOOLQ PLFURJUDPV SHU POf DQG EURPRFKORURLQGRO\O JDODFWRS\UDQRVLGH ;*DOf PLFURJUDPV SHU POf DQG LQFXEDWHG IRU 2 2 WR KRXUV DW & $OO WUDQVIRUPDQWV ZHUH VWRUHG DW & LQ /% EURWK ZWK DPSLFLOOLQ PLFURJUDPV SHU POf DQG b JO\FHURO 3UHSDUDWLRQ RI $QWLVHUD /DWH H[SRQHQWLDO SKDVH FHOOV RI % JLQJLYDOLV VWUDLQ ZHUH SHOOHWHG ZDVKHG ZWK 0 SKRVSKDWHEXIIHUHG VDOLQH 3%6f S+ 2 DQG UHVXVSHQGHG LQ 3%6 DQG b VRGLXP D]LGH DW & IRU DW OHDVW KRXU 7KH FHOOV ZHUH DJDLQ ZDVKHG ZLWK 3%6 UHVXVSHQGHG WR D FRQFHQWUDWLRQ RI [ FHOOV SHU PO DQG HPXOVLILHG LQ DQ HTXDO YROXPH RI )UHXQGnV LQFRPSOHWH DGMXYDQW 7KH FHOO HPXOVLRQ ZDV LQMHFWHG LQ GRVHV DW WZR ZUHHN LQWHUYDOV IRU ZHHNV VXEFXWDQHRXVO\ LQ WKH EDFN RI DGXOW 1HZ =HDODQG UDEELWV (DFK UDEELW ZDV JLYHQ D ERRVWHU GRVH WR GD\V ODWHU $QWLVHUD ZfHUH FROOHFWHG IURP WKH PDUJLQDO HDU YHLQV MXVW SULRU WR LPPXQL]DWLRQ DQG EHJLQQLQJ RQH ZHHN DIWHU WKH ERRVWHU 2 GRVH $OO VHUD ZHUH VWRUHG DW & 5DEELW DQWL% JLQJLYDOLV DQWLVHUXP ZDV DGVRUEHG WLPHV ZWK ( FROL -0 KDUERULQJ S8& SODVPLG ( FROL -0 S8& f )RU HDFK DGVRUSWLRQ ( FROL FHOOV IURP OLWHU RI D VWDWLRQDU\ SKDVH FXOWXUH 2 ZHUH ZDVKHG DQG PL[HG ZLWK PO RI VHUXP DW & IRU KRXU 7KH VHUXP ZDV UHFRYHUHG E\ SHOOHWLQJ WKH FHOOV DW [ J IRU PLQXWHV )RU VRQLFDWH DGVRUSWLRQ ( FROL FHOOV IURP PO RI VWDWLRQDU\ SKDVH JURZWK VXVSHQGHG LQ PO RI 3%6 ZHUH GLVUXSWHG E\ VRQLFDWLRQ DQG PL[HG ZLWK ( 2 FROL FHOODGVRUEHG VHUXP IRU KRXU DW & 7KH PL[WXUH ZDV FHQWULIXJHG

PAGE 29

DW [ J IRU KRXU DQG WKH UHVXOWLQJ GHDU VHUXP ZDV VWRUHG DW 2 & $VVD\ RI $QWLERG\ 7LWHU 6HUD ZHUH WHVWHG IRU DQWL JLQJLYDOLV DQG DQWL( FROO DFWLYLWLHV E\ DQ HQ]\PHOLQNHG LPPXQRVRUEHQW DVVD\ (/,6$f % JLQJLYDOLV FHOOV VXVSHQGHG LQ FDUERQDWHELFDUERQDWH EXIIHU S+ FHOOV 2 SHU ZHOOf ZHUH IL[HG WR PLFURWLWHU SODWHV DW & RYHUQLJKW $IWHU WKH ZHOOV ZHUH ZDVKHG ZLWK b 7ZHHQ LQ 3%6 b ERYLQH VHUXP DOEXPLQ %6$f LQ 3%6 ZDV DGGHG WR HDFK ZHOO DQG WKH SODWHV ZHUH LQFXEDWHG IRU KRXUV DW URRP WHPSHUDWXUH LQ RUGHU WR VDWXUDWH WKH ELQGLQJ VLWHV $IWHU ZDVKLQJ WKH SODWHV VHULDOO\ GLOXWHG DQWLVHUXP ZDV DGGHG DQG SODWHV ZHUH LQFXEDWHG IRU KRXU DW URRP WHPSHUDWXUH IROORZHG E\ D VHFRQG ZDVK ZLWK b 7ZHHQ LQ 3%6 3HUR[LGDVH FRQMXJDWHG JRDW DQWLUDEELW ,J* GLOXWHG LQ b %6$ Z"DV DGGHG DQG WKH SODWHV ZHUH DJDLQ LQFXEDWHG DW URRP WHPSHUDWXUH IRU KRXU $IWHU D ILQDO ZDVKLQJ D FRORUIRUPLQJ VXEVWUDWH VROXWLRQ 2SKHQ\OHQHGLDPLQH J SHU PO LQ 0 FLWUDWH EXIIHU S+ DQG b K\GURJHQ SHUR[LGHf ZDV DGGHG DQG WKH SODWHV ZHUH LQFXEDWHG IRU PLQXWHV DW URRP WHPSHUDWXUH 7KH DEVRUEDQFH DW QP ZUDV PHDVXUHG ZLWK D 7LWHUWHN 0XOWLVFDQ UHDGHU $Q DEVRUEDQFH RI RU PRUH RYHU EDFNJURXQG ZDV FRQVLGHUHG SRVLWLYH %DFNJURXQG UHDGLQJV ZHUH REWDLQHG IURP WKH ZHOOV LQ ZKLFK DOO UHDJHQWV H[FHSW DQWL JLQJLYDOLV DQWLVHUXP ZDV DGGHG 1RUPDO UDEELW VHUXP ZDV DOVR WHVWHG DJDLQVW % JLQJLYDOLV DQWLJHQ 7R WHVW WKH HIIHFWLYHQHVV RI DGVRUSWLRQ WKH WLWHUV RI WUHDWHG VHUD ZnHUH DVVD\HG DV GHVFULEHG DERYH H[FHSW WKDW ( FROL -0 S8&f ZKROH FHOOV ZHUH XVHG DV WKH DQWLJHQ

PAGE 30

)LOWHUf§%LQGLQJ (Q]\PH LPPXQRDVVD\ $PSLFLOOLQUHVLVWDQW WUDQVIRUPDQWV ZKLFK IRUPHG ZKLWH FRORQLHV LQ WKH SUHVHQFH RI ;*DO ZHUH VSRWWHG RQWR /% DJDU SODWHV ZLWK DUQSLFLOOLQ JURZQ RYHUQLJKW DQG EORWWHG RQWR QLWURFHOOXORVH ILOWHU GLVNV % JLQJLYDOLV DQG ( FROL -0 S8& f ZHUH DOVR VSRWWHG RQWR HDFK ILOWHU DV D SRVLWLYH DQG QHJDWLYH FRQWURO UHVSHFWLYHO\ 'XSOLFDWH SULQWV RI WKH FRORQLHV RQ QLWURFHOOXORVH ILOWHUV ZHUH PDGH DQG FRORQLHV RQ RQH RI HDFK GXSOLFDWH SULQW ZHUH O\VHG E\ D PLQ H[SRVXUH WR FKORURIRUP YDSRU )LOWHUV ZHUH WKHQ DLU GULHG IRU PLQXWHV DQG VRDNHG IRU KRXUV LQ 3%6 FRQWDLQLQJ b ERYLQH VHUXP DOEXPLQ $IWHU WKH ILOWHUV ZHUH XUDVKHG DGVRUEHG UDEELW DQWL JLQJLYDOLV DQWLVHUXP WSDV DGGHG DQG WKH ILOWHUV ZHUH LQFXEDWHG LQ D VROXWLRQ RI SHUR[LGDVH FRQMXJDWHG JRDW DQWLUDEELW LPPXQRJOREXOLQ IRU KRXU $IWHU ZDVKLQJ WKH ILOWHUV ZHUH GHYHORSHG LQ D FRORUIRUPLQJ VXEVWUDWH VROXWLRQ FRQVLVWLQJ RI b FKORURO QDSKWKRO DQG b K\GURJHQ SHUR[LGH LQ D VROXWLRQ RI PHWKDQRO7%6 P0 7ULV K\GURFKORULGH P0 1D&O S+ f &ORQHV ZKLFK GHYHORSHG D EOXH FRORU ZHUH SLFNHG DQG UHVFUHHQHG E\ WKH VDPH SURFHGXUH 5HVWULFWLRQ $QDO\VLV RI 5HFRPELQDQW 3ODVPLGV 3ODVPLGV ZHUH LVRODWHG IURP DOO WKH FORQHV WKDW ZHUH SRVLWLYH LQ WKH ILOWHUELQGLQJ HQ]\PH LPPXQRDVVD\ 5HVWULFWLRQ HQGRQXFOHDVH GLJHVWLRQV ZHUH SHUIRUPHG XQGHU FRQGLWLRQV GHVFULEHG E\ WKH PDQXIDFWXUHU WR SURGXFH FRPSOHWH GLJHVWLRQ $JDURVH JHO HOHFWURSKRUHVLV ZDV SHUIRUPHG DV GHVFULEHG E\ 0DQLDWLV HW DO f 7KH VL]H RI '1$ EDQGV ZDV HVWLPDWHG E\ FRPSDULQJ WKH GLVWDQFH RI PLJUDWLRQ WR D ORJULWKPLF SORW RI WKH PLJUDWLRQ RI VWDQGDUG UHVWULFWHG

PAGE 31

ODPEGD '1$ UXQ RQ WKH VDPH JHO 6RXWKHUQ 3ORW $QDO\VLV 5HFRPELQDQW SODVPLG DQG S8& YHFWRU '1$V ZHUH GLJHVWHG WR FRPSOHWLRQ ZLWK WKH DSSURSULDWH UHVWULFWLRQ HQ]\PHV DQG UXQ RQ D b DJDURVH JHO % JLQJLYDOLV '1$ SDUWLDOO\ GLJHVWHG ZLWK 6DX $ DQG +LQG ,,, GLJHVWHG (LNHQHOOD FRUURGHQV FORQH '1$ XQSXEOLVKHGf ZHUH DOVR ORDGHG LQ WKH JHO 7KH '1$ ZDV WUDQVIHUUHG WR %LRG\QH Q\ORQ PHPEUDQH E\ 6RXWKHUQ WUDQVIHU 6RXWKHUQ f % JLQJLYDOLV '1$ SDUWLDOO\ GLJHVWHG ZLWK +LQG ,,, ZDV QLFN WUDQVODWHG ZLWK D 3 G&73f &LPPRO $PHUVKDP &RUS $UOLQJWRQ +HLJKWV ,OOf DV GHVFULEHG E\ 0DQLDWLV HW DO f 7KH PHPEUDQHERXQG '1$ ZnDV 2 K\EULGL]HG WR WKH QLFNWUDQVODWHG SUREH DW & LQ b IRUPDPLGH IRU KRXUV E\ WKH PHWKRG UHFRPPHQGHG E\ WKH PDQXIDFWXUHU 3DOO 8OWUDILQH )LOWUDWLRQ &RUS *OHQ &RYH 1
PAGE 32

FXOWXUHV DW D ILQDO FRQFHQWUDWLRQ RI UQ0 DQG WKH FHOV ZHUH SHOOHWHG E\ FHQWULIXJDWLRQ KRXUV ODWHU 7KH FHOOV ZHUH ZDVKHG UHVXVSHQGHG LQ YROXPH RI 3%6 DQG WKH RSWLFDO GHQVLW\ RI HDFK VXVSHQVLRQ ZDV GHWHUPLQHG DW ULP &HOO O\VDWH DQWLJHQ ZDV SUHSDUHG E\ EUHDNLQJ WKH FHOOV ZLWK D VRQLFDWRU 7KH SURWHLQ FRQFHQWUDWLRQ RI HDFK O\VDWH ZDV GHWHUPLQHG E\ WKH %LR5DG SURWHLQ DVVD\ %LR5DG /DERUDWRULHV 5LFKPRQG &DOLIf 'HWHUPLQDWLRQ RI WKH WLWHU RI DQWL JLQJLYDOLV DJDLQVW WKHVH DQWLJHQV ZDV SHUIRUPHG ZLWK WKH (/,6$ DV GHVFULEHG DERYH % FHOOV RU SJ SURWHLQ SHU ZHOOf 1RUPDO UDEELW VHUXP H[KDXVWLYHO\ DGVRUEHG ZLWK ( FROL -0 S8&f ZDV DOVR WHVWHG LQ WKH VDPH PDQQHU 6RGLXP 'RGHF\O 6XOIDWH3RO\DFU\ODPLGH *HO (OHFWURSKRUHVLV 6'63$*(f DQG :HVWHUQ %ORW $QDO\VLV RI WKH ([SUHVVHG $QWLJHQV (DFK RI WKH UHSUHVHQWDWLYH DQWLJHQSURGXFLQJ FORQHV ZDV JURZQ WR PLGORJ SKDVH LQ PO RI /% EURWK ZLWK PLFURJUDPV RI DPSLFLOOLQ SHU PO 7KH FHOOV ZHUH SHOOHWHG ZDVKHG ZLWK 3%6 UHVXVSHQGHG LQ PO RI VDPSOH EXIIHU P0 7ULVK\GURFKORULGH b PHUFDSWRHWKDQRO b 6'6 b JO\FHURO b EURPRSKHQRO EOXH S+ f DQG ERLOHG IRU PLQXWHV 7KH % JLQJLYDOLV FHOO O\VDWH ZDV PL[HG ZLWK DQ HTXDO YROXPH RI VDPSOH EXIIHU DQG WUHDWHG LQ WKH VDPH PDQQHU 6'63$*( ZDV SHUIRUPHG LQ D YHUWLFDO VODE JHO HOHFWURSKRUHVLV WDQN +RHIHU 6FLHQWLILF ,QVWUXPHQWV 6DQ )UDQFLVFR &$f DV GHVFULEHG E\ /DHPPOL f 6DPSOHV RI PO IURP HDFK FORQH DV ZHOO DV PLFURJUDPV RI %DFWHURLGHV FHOO O\VDWH ZHUH UXQ DW D FRQVWDQW FXUUHQW RI P$ SHU JHO WKURXJK WKH b SRO\DFU\ODPLGH VWDFNLQJ JHO S+ f DQG P$ SHU JHO WKURXJK WKH b RU b VHSDUDWLQJ JHO S+ f 7KH JHOV ZHUH

PAGE 33

SURFHVVHG HLWKHU E\ VWDLQLQJ ZLWK &RRPDVVLH EULOOLDQW EOXH 5 )DLUEDQNV HW DO f RU XVHG IRU :HVWHUQ EORW DQDO\VLV :HVWHUQ EORWWLQJ ZDV GRQH DV GHVFULEHG E\ %XUQHWWH f DV IROORZV 6HSDUDWHG DQWLJHQV RQ WKH JHO ZHUH WUDQVIHUUHG WR QLWURFHOOXORVH SDSHU Pf 6FKOHLFKHU t 6FKXHOO &R ,QF .HHQH 1+f E\ HOHFWUREORWWLULJ XVLQJ WKH +RHIHU DSSDUDWXV DW &2 9 RYHUQLJKW ZLWK D EXIIHU FRQWDLQLQJ PLOO 7ULV EDVH P0 JO\FLQH DQG b PHWKDQRO S+ f 7KH EORW ZDV YLVXDOL]HG DV IROORZV 1LWURFHOOXORVH VKHHWV ZHUH SUHLQFXEDWHG LQ D EORFNLQJ VROXWLRQ RI 3%6 ZLWK b %6$ DQG b 7ZHHQ IRU KRXUV RU RYHUQLJKW $GVRUEHG DQWLVHUD XVHG DV SUREHV ZHUH XVXDOO\ GLOXWHG LQ EORFNLQJ VROXWLRQ DQG UHDFWHG ZLWK WKH QLWURFHOOXORVH WUDQVIHU IRU KRXUV $IWHU Zn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f ZKHUHDV QRUPDO UDEELW VHUXP KDG DQ DQWLERG\ WLWHU RI WR % JLQJLYDOLV DQG WR ( FROL S8& f $GVRUSWLRQ RI DQWL% JLQJLYDOLV DQWLVHUXP ZLWK ( FROL S8& f UHVXOWHG LQ D VOLJKW UHGXFWLRQ RI DQWLERG\ WLWHU

PAGE 34

WR % JLQJLYDOLV DQG UHGXFHG WKH DQWLVn FROL WLWHU WR ]HUR RU ,GHQWLILFDWLRQ RI ( FROL 7UDcVIRUPDQWV :KLFK ([SUHVVHG % JLQJLYDOLV $QWLJHQV $SSUR[LPDWHO\ WUDQIRUPDQWV JHQHUDWHG IURP WKH 6DX $ UHVWULFWHG FKURPRVRPDO '1$ ZHUH WHVWHG IRU WKH H[SUHVVLRQ RI % JLQJLYDOLV DQWLJHQV E\ WKH ILOWHUELQGLQJ HQ]\PH LPPXQRDVVD\ XVLQJ ( FROL DGVRUEHG UDEELW DQWL% JLQJLYDOLV VHUXP 2QO\ FORQH FORQH f ZDV SRVLWLYH ZKHQ HLWKHU O\VHG RU XQO\VHG FHOOV ZHUH WHVWHG $ WRWDO RI FRORQLHV RI WUDQVIRUPDQWV UHVXOWLQJ IURP +LQG ,,, UHVWULFWHG FKURPRVRPDO '1$ ZHUH DOVR WHVWHG IRU WKH H[SUHVVLRQ RI % JLQJLYDOLV DQWLJHQV 6HYHQ FORQHV JDYH SRVLWLYH VLJQDOV 2I WKHVH FORQHV RQH X7DV SRVLWLYH RQO\ ZKHQ O\VHG FORQH f DQG WKH UHVW ZHUH SRVLWLYH ERWK ZKHQ O\VHG DQG XQO\VHG 7DEOH f $JDURVH *HO (OHFWURSKRUHVLV RI 5HFRPELQDQW 3ODVPLGV 7R IXUWKHU FRQILUP WKH SRVLWLYH UHVXOWV RI WKH ILOWHUELQGLQJ HQ]\PH LPPXQRDVVD\ SODVPLG '1$ ZDV LVRODWHG IURP HDFK SRVLWLYH FORQH (OHFWURSKRUHVLV RI WKHVH XQUHVWULFWHG SODVPLGV VKRZHG WKDW HDFK FORQH FRQWDLQHG RQO\ RQH UHFRPELQDQW SODVPLG )LJXUH ODQHV WKURXJK f &ORQH ZKLFK ZDV FRQVWUXFWHG E\ OLJDWLRQ RI 6DX $ SDUWLDOO\ GLJHVWHG % JLQJLYDOLV '1$ ZLWK %DP +, FXW S8& FRXOG QRW EH GLJHVWHG ZLWK %DP +, )LJXUH ODQH f 5HVWULFWLRQ RI S8& ZLWK HQ]\PH 6PD DQG 6DO GHOHWHV D ES IUDJPHQW FRQWDLQLQJ WKH %DP +, VLWH IURP S8& )LJXUH ODQH DQG )LJXUH ODQH VHH )LJXUH IRU PDS RI S8& f 7KHUHIRUH FORQH '1$ ZDV UHVWULFWHG ZLWK 6PD DQG 6DO 5HVWULFWLRQ DQDO\VLV UHYHDOHG D IUDJPHQW RI OLQHDU ESGHOHWHG S8&

PAGE 35

7DEOH &KDUDFWHUL]DWLRQ RI ( FROL WUDQVIRUPDQWV ZKLFK H[SUHVV % JLQJLYDOLV DQWLJHQV &RORQLHV UHDFWHG 6L]H RI % &ORQH 1R ZLWK DQWLVHUXP XQO\VHG O\VHG JLQJLYDOLV '1$ FORQHG .Ef DQG D DQG f§ E D 3RVLWLYH UHDFWLRQ E 1HJDWLYH QRW UHDFWLYH

PAGE 36

)LJXUH $JDURVH JHO HOHFWURSKRUHVLV RI UHFRPELQDQW SODVPLGV /DQHV XQGLJHVWHG UHFRPELQDQW SODVPLGV IURP FORQHV S8& GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLGV IURP FORQHV DQG GLJHVWHG ZLWK +LQG ,,, S8& GLJHVWHG ZLWK 3YX ,, UHFRPELQDQW SODVPLGV RI FORQHV DQG GLJHVWHG ZLWK 3YX ,, S8& GLJHVWHG ZLWK 6PD DQG 6DO UHFRPELQDQW SODVPLG RI FORQH GLJHVWHG ZLWK 6PD DQG 6DO ,

PAGE 38

DQG IUDJPHQWV RI LQVHUW )LJXUH ODQH DQG )LJXUH ODQH f 5HVWULFWLRQ DQDO\VLV ZLWK GLIIHUHQW HQ]\PHV )LJXUH f VKRZHG WKDW WKH VL]H RI LQVHUW RI FORQH ZDV DSSUR[LPDWHO\ NE &ORQHV DQG ZHUH JHQHUDWHG IURP +LQG ,OO UHVWULFWHG FKURPRVRPDO '1$ $IWHU GLJHVWLRQ ZLWK +LQG ,,, RQO\ FORQHV DQG UHYHDOHG IUDJPHQWV RI WKH OLQHDU S8& YHFWRU DQG IUDJPHQWV RI % JLQJLYD-LV '1$ LQVHUWV )LJXUH ODQHV WKURXJK f 3ODVPLG '1$V RI WKHVH FORQHV ZHUH UHVWULFWHG ZLWK YDULRXV HQ]\PHV DQG DQDO\]HG E\ JHO HOHFWURSKRUHVLV )LJXUH f 7KH HVWLPDWHG VL] RI LQVHUWV RI FORQHV DQG DUH DQG NE UHVSHFWLYHO\ 7DEOH f 7KXV FORQHV DQG ZHUH IRXQG WR FRQWDLQ SODVPLGV RI WKH VDPH VL]H DQG LGHQWLFDO UHVWULFWLRQ IUDJPHQWV $OWKRXJK FORQHV DQG ZHUH JHQHUDWHG IURP +LQG ,,, UHVWULFWHG '1$ WKH\ GLG QRW UHVXOW LQ IUDJPHQWV RI OLQHDU S8& DIWHU +LQG ,,, GLJHVWLRQ )LJXUH ODQHV DQG f 7KHVH FORQHG '1$V ZHUH WKHQ UHVWULFWHG ZLWK 3YX ,, ZKLFK JHQHUDWHV D ES IUDJPHQW FRQWDLQLQJ WKH SRO\OLQNHUFORQLQJ VLWHV IURP S8& )LJXUH DQG )LJXUH ODQH DQG )LJXUH ODQH f &ORQHV DQG UHYHDOHG IUDJPHQWV RI OLQHDU ESGHOHWHG S8& DQG LQVHUWV DVVRFLDWHG ZLWK WKH GHOHWHG IUDJPHQW )LJXUH ODQHV DQG f 7KHVH FORQHG '1$V ZHUH GLJHVWHG ZGWK YDULRXV UHVWULFWLRQ HQ]\PHV DQG DQDO\]HG E\ DJDURVH JHO HOHFWURSKRUHVLV )LJXUH f 7KH VL]H RI LQVHUWV RI FORQHV DQG ZHUH HVWLPDWHG WR EH DQG NE UHVSHFWLYHO\ 7DEOH f &ORQHV DQG DOVR FRQWDLQHG SODVPLGV RI WKH VDPH VL]H DQG LGHQWLFDO UHVWULFWLRQ IUDJPHQWV

PAGE 39

)LJXUH $JDURVH JHO HOHFWURSKRUHVLV RI GLIIHUHQW UHVWULFWLRQ GLJHVWV RI WKH UHFRPELQDQW SODVPLG IURP FORQH /DQHV '1$ PDUNHU+LQG (FR 5, GLJHVW RI ODPEGD '1$ XQGLJHVWHG S8& S8& GLJHVWHG ZLWK +LQG ,,, S8& GLJHVWHG ZLWK 6PD DQG 6DO DQG UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 6PD DQG 6DO 6PD DORQH 6DO DORQH +LQG ,,, (FR 5, DQG %DP +, UHVSHFWLYHO\ XQGLJHVWHG UHFRPELQDQW SODVPLG IURP FORQH '1$ PDUNHU+LQG ,,, GLJHVW RI ODPEGD '1$

PAGE 41

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

PAGE 43

)LJXUH $JDURVH JHO HOHFWURSKRUHVLV RI GLIIHUHQW UHVWULFWLRQ GLJHVWV RI UHFRPELQDQW SODVPLGV IURP FORQHV DQG /DQHV '1$ PDUNHU+LQG ,,,(FR 5, GLJHVW RI ODPEGD '1$ S8& XQGLJHVWHG S8& GLJHVWHG ZLWK +LQG ,,, S8& GLJHVWHG ZLWK 3YX ,, DQG UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 3YX ,, +LQG ,,, (FR 5, DQG %DP +, UHVSHFWLYHO\ XQGLJHVWHG UHFRPELQDQW SODVPLG IURP FORQH DQG UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 3YX ,, +LQG ,,, (FR 5, DQG %DP +, UHVSHFWLYHO\ XQGLJHVWHG UHFRPELQDQW SODVPLG IURP FORQH DQG UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 3YX ,, +LQG ,,, (FR 5, DQG %DP +, UHVSHFWLYHO\ XQGLJHVWHG UHFRPELQDQW SODVPLG RI FORQH

PAGE 45

+\EULG QDWLRQ RI 5HFRPELQDQW 3ODVPLGV ULWK JLQJLYDOLV ';$ 3UREH 6RXWKHUQ W LRW DQDO\VLV ZDV DOVR SHUIRUPHG WR FRQILUP WKDW WKH '1$ LQVHUWV ZHUH GHULYHG IURP WKH 3 JLQJLYDOLV '1$ $V FDQ EH VHHQ LQ )LJXUH WKH K\EULGL]DWLRQ SDWWHUQ RI PRVW RI WKH LQVHUW IUDJPHQWV VKRZHG GDUN EDQGV RI KRPRORJ\ WR WKH % JLQJLYDOLV FKURPRVRPDO '1$ SUREH 7KH S8& VKRZHG D IDLQW EDQG ZLWK KRPRORJ\ WR WKH SUREH ,QFUHDVLQJ WKH VWULQJHQF\ RI WKH ZDVK r & IRU KRXUf GLG QRW VLJQLILFDQWO\ FKDQJH WKH K\EULGL]DWLRQ SDWWHUQ +RZnHYHU D VKRUWHU H[SRVXUH RI WKH DXWRUDGLRJUDSK HOLPLQDWHG WKH EDFNJURXQG RI S8& EXW WKH WZR VPDOOHVW LQVHUW EDQGV IURP FORQH DOVR GLVDSSHDUHG 7KH FRQWURO '1$ IURP (LNHQHOOD FRUURGHQV GLG QRW K\EULGL]H ZLWK WKH 3 JLQJLYDOLV '1$ SUREH )LJXUH ODQH f 7LWHU RI $QWLIO JLQJLYDOLV $QWLVHUXP WR ( FROL 7UDQVIRUPDQWV $QWL% JLQJLYDOLV DQWLVHUXP ZDV DEOH WR GHWHFW DQWLJHQ H[SUHVVLRQ LQ DOO SRVLWLYH FORQHV H[FHSW FORQH LQ DQ HQ]\PHOLQNHG LPPXQRVRUEHQW DVVD\ (/,6$f 7DEOH f 7KH DQWLVHUXP UHDFWHG ZLWK ERWK ZKROH FHOO DQG FHOO O\VDWH DQWLJHQV ,VRSURS\O 'WKLRJDODFWRS\UDQRVLGH ,37*f ZDV QRW QHFHVDU\ WR LQGXFH DQWLJHQ H[SUHVVLRQ +RZHYHU LQ WKH SUHVHQFH RI ,37* FORQHV DQG VKRZHG KLJKHU DQWLJHQ H[SUHVVLRQ HVSHFLDOO\ ZKHQ WKH FHOO O\VDWH SUHSDUDWLRQV ZHUH WHVWHG 'HWHUPLQDWLRQ RI WKH ([SUHVVHG $QWLJHQV LQ ( FROL -0 )LYH VWDEOH UHSUHVHQWDWLYH FORQHV ZHUH DQDO\]HG IRU DQWLJHQ H[SUHVVLRQ E\ 6'63$*( DQG :HVWHUQ EORW DQDO\VLV $V FDQ EH VHHQ LQ )LJXUH RQO\ FORQHV DQG SURGXFHG DQWLJHQV GHWHFWDEOH E\ ( FROL DGVRUEHG DQWLIK JLQJLYDOLV DQWLVHUXP LQ WKH :HVWHUQ EORW $QWLJHQV

PAGE 46

)LJXUH +\EULGL]DWLRQ RI UHFRPELQDQW SODVPLGV ZLWK 3 ODEHOHG % JLQJLYDOLV '1$ SUREH $ $JDURVH JHO HOHFWURSKRUHVLV RI '1$ EHIRUH 6RXWKHUQ WUDQVIHU /DQHV 6DX $ SDUWLDOO\ GLJHVWHG % JLQJLYDOLV '1$ S8& GLJHVWHG ZLWK 3YX ,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 3YX ,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 3YX ,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 3YX ,, S8& GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK 6PD DQG 6DO DQG UHFRPELQDQW SODVPLG IURP (LNHQHOOD FRUYRGHQV FORQH GLJHVWHG ZLWK +LQG ,,, % $XWRUDGLRJUDSK RI 6RXWKHUQ EORW K\EULGL]DWLRQ RI WKH DJDURVH JHO LQ SDQHO $

PAGE 48

7DEOH 7LWHU RI DQWL JLQJLYDOLV DQWLVHUXP DJDLQVW ( FROL WUDQVIRUPDQWV ZKLFK H[SUHVV %JLQJLYDOLV DQWLJHQV $QWLERG\ WLWHUVp DJDLQVW WHVW DQWLJHQVE 2UJDQLVP :KROH FHOO &HOO O\VDWH ,37* ,37* ,37* ,37* &ORQH 17& 17 17 17 17 ( FROL -0 S8& f % JLQJLYDOLV 17 17 17 &RQWURO 156G D 1XPEHU GHVLJQDWHV WKH UHFLSURFDO GLOXWLRQ RI WKH VHUD ZKLFK JDYH 2' UHDGLQJ RI RU PRUH RYHU WKH EDFNJURXQG $QWLVHUXP ZDV H[KDXVWLYHO\ DGVRUEHG ZLWK ( FROL -0 S8& f E $QWLJHQV ZHUH SUHSDUHG IURP FXOWXUHV JURZQ ZLWKRXW ,37* ,37*af RU LQ WKH SUHVHQFH RI ,37* ,37*! F 1RW WHVWHG G 1RUPDO UDEELW VHUXP H[KDXVWLYHO\ DGVRUEHG ZLWK ( FROL -0 S8& f GLG QRW UHDFW WR WHVW DQWLJHQV

PAGE 49

)LJXUH 6'63$*( RQ b DFU\ODPLGH DQG :HVWHUQ EORW DQDO\VLV RI H[SUHVVHG % JLQJLYDOLV DQWLJHQV 0ROHFXODU ZHLJKW VWDQGDUGV 3KDUPDFLD )LQH &KHPLFDOV 3LVFDWDZD\ 1
PAGE 51

R H[SUHVVHG LQ FORQHV DQG ZHUH QRW GHWHFWHG E\ :HVWHUQ EORW DQDO\VLV 1RUPDO UDEELW VHUXP UHDFWHG WR VRPH FRPPRQ DQWLJHQV DPRQJ WKHVH FORQHV DQG ( FROL -0 S8& f 7KH DQWL% JLQJMYDOLV DQWLVHUXP GLG KRZHYHU UHDFW ZLWK D SURWHLQ EDQG RI DSSUR[LPDWHO\ .f DV ZHOO DV D VPHDU RI ORZHU PROHFXODU ZHLJKW IURP FORQH 0XOWLSOH EDQGV RI WR IURP FORQH ZHUH DOVR GHWHFWHG 7KHVH SDUWLFXODU SRO\SHSWLGHV ZHUH QRW GHWHFWDEOH LQ ( FROL -0 S8& f SUHSDUDWLRQV )LJXUH ODQH f $ ZKROH FHOO SUHSDUDWLRQ IURP FORQH ZDV DOVR VHSDUDWHG LQ D b 6'6 SRO\DFU\ODPLGH JHO DQG WKH H[SUHVVHG SURWHLQ ZnDV HVWLPDWHG WR KDYH D PROHFXODU XnHLJKW RI )LJXUH f 'LVFXVVLRQ *HQRPLF OLEUDULHV RI % JLQJLYDOLV '1$ ZHUH FRQVWUXFWHG LQ WKH SODVPLG H[SUHVVLRQ YHFWRU S8& ZKLFK FRQWDLQV WKH S%5 RULJLQ RI UHSOLFDWLRQ WKH S%5 DPSLFLOOLQ UHVLVWDQFH JHQH DQG D SRUWLRQ RI WKH ODF = JHQH RI ( FROL ZKLFK FRGHV IRU WKH D SHSWLGH RI JDODFWRVLGDVH )LJXUH f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nKLWH FRORQLHV RQ ;*DO FRQWDLQLQJ SODWHV 7KH DGYDQWDJHV WR WKLV SODVPLG DUH f '1$ LQVHUWHG

PAGE 52

)LJXUH 6'63$*( RQ b DFU\ODPLGHf RI H[SUHVVHG % JLQJLYDOLV DQWLJHQ LQ FORQH /DQHV $f 0ROHFXODU ZHLJKW VWDQGDUGV 6LJPD &KHPLFDO &R 6W /RXLV 0Rf DUH P\RVLQ f JJDODFWRVLGDVH f SKRVSKRU\ODVH % f %f :KROH FHOO VDPSOH RI FORQH

PAGE 53

.

PAGE 54

LQWR DQ\ RI WKH FORQLQJ VLWHV ZKLFK DUH GRZQVWUHDP IURP D VWURQJ SURPRWHU VKRXOG EH H[SUHVVHG ZKHWKHU RU QRW D % JLQJLYDOOV SURPRWHU LV FORQHG ZLWK D VWUXFWXUDO JHQH f WUDQVIRUPDQWV FRQWDLQLQJ D UHFRPELQDQW SODVPLG DUH HDVLO\ GHWHFWHG XSRQ LQLWLDO VHOHFWLRQ DQG f WKH PXOWLSOH FORQLQJ VLWHV PDNH LW D YHUVDWLOH FORQLQJ YHFWRU ZKLFK LV HVSHFLDOO\ XVHIXO IRU VXEFORQLQJ )LYH GLIIHUHQW ( FRLL FORQHV VWDEO\ H[KLELWHG % JLQJLYDLLV DQWLJHQ H[SUHVVLRQ 7KHVH DQWLJHQV ZHUH GHWHFWHG LQ LQWDFW FHOOV ERWK E\ ILOWHUELQGLQJ HQ]\PH LPPXQRDVVD\ 7DEOH f DQG (/,6$ 7DEOH f $OWKRXJK LW KDV QRW \HW EHHQ FRQILUPHG E\ LPPXQRHOHHWURQPLFURVFRS\ LW LV OLNHO\ WKDW WKHVH %DFWHURLGHV DQWLJHQV DUH ORFDWHG RQ WKH ( FROL FHOO VXUIDFH DQG WKHUHIRUH PXVW FRQWDLQ D OHDGHU SHSWLGH LQ RUGHU WR EH WUDQVORFDWHG WR WKH ( FROL VXUIDFH 2OLYHU f 7KLV UHVXOW VXJJHVWV WKDW % JLQJLYDLLV VXUIDFH DQWLJHQV FDQ EH SURFHVVHG DV ZHOO DV H[SUHVVHG LQ ( FROL &ORQHV DQG KDYH XQGHUJRQH VRPH NLQG RI '1$ UHDUUDQJHPHQW LH WKH UHFRPELQDQW SODVPLGV nZKHQ FXW E\ +LQG ,,, GLG QRW UHVXOW S8& DQG LQVHUW EDQG EXW VKRZHG RQH ODUJH EDQG DQG RQH VPDOO EDQG )LJXUH ODQHV DQG f 7KLV DSSDUHQW UHDUUDQJHPHQW PD\ UHVXOW IURP D GHOHWLRQ DW RQH +LQG ,,, HQG RI WKH LQVHUW DQG DQRWKHU +LQG ,,, HQG PD\ VWLOO EH LQWDFW &ORQH ZDV IRXQG WR HQFRGH D SRO\SHSWLGH ZLWK DQ DYHUDJH PROHFXODU ZHLJKW RI VHHQ LQ SRO\DFU\ODPLGH JHOV DQG GHWHFWHG E\ :HVWHUQ EORW DQDO\VLV )LJXUHV DQG f 7KH VPHDU DW WKH ORZHU PROHFXODU ZHLJKW VHHQ LQ WKH EORW PD\ EH WKH GHJUDGHG SURGXFW RI WKLV H[SUHVVHG DQWLJHQ VLQFH ( FROL KDV D IXQFWLRQDO ,RQ JHQH ZKLFK HQFRGHV IRU WKH HQ]\PH LQYROYHG LQ GHJUDGDWLRQ RI LQWHUQDO DEQRUPDO

PAGE 55

SURWHLQV &KDUHWWH HW DO &KXQJ DQG *ROGEHUJ :D[PDQ DQG *ROGEHUJ f 7KH IXQFWLRQ RI WKH ODF SURPRWHU LQ S8& GRHV QRW GHSHQG RQ ,37* LW LV KRZHYHU HQKDQFHG E\ ,37* VLQFH ( FROL -0 S8& f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fKLFK ZRXOG DGG DSSUR[LPDWHO\ DPLQR DFLGV WR WKH H[SUHVVHG SRO\SHSWLGH 7KH H[SUHVVLRQ RI WKH FORQH DQWLJHQ ZDV DOVR IRXQG WR EH VWLPXODWHG E\ ,37* LQ WKH VDPH PDQQHU DV FORQH 7KH VL]H RI WKH FORQH LQVHUW ESf LV ODUJH HQRXJK WR HQFRGH IRU WKH H[SUHVVHG DQWLJHQ WR .f REVHUYHG E\ :HVWHUQ EORWWLQJ 7KH V\QWKHVLV RI %DFWHURLGHV DQWLJHQV LQ FORQHV DQG ZDV QRW IRXQG WR GHSHQG RQ WKH SUHVHQFH RI ,37* RU WR EH HQKDQFHG E\ ,37* 7DEOH f 7KLV VXJJHVWV WKDW D IXQFWLRQDO %DFWHURLGHV SURPRWHU LV LQFOXGHG ZLWK WKH VWUXFWXUDO JHQH RI HDFK FORQH +RZHYHU DQWLJHQ H[SUHVVLRQ RI WKHVH FORQHV FDQQRW EH GHWHFWHG E\ :HVWHUQ EORW DQDO\VLV 7KLV PLJKW EH GXH WR f WKH DQWLJHQV QRW EHLQJ WUDQVIHUUHG WR WKH QLWURFHOOXORVH VKHHWV f WKH WUDQVIHUUHG DQWLJHQV FRQWDLQLQJ DOWHUHG FRQIRUPDWLRQV ZKLFK DUH QRW UHFRJQL]HG E\ WKH DQWLVHUXP RU f WKH DQWLJHQ H[SUHVVLRQ EHLQJ WRR ORZ WR EH GHWHFWHG

PAGE 56

7KHVH UHVXOWV KDYH GHPRQVWUDWHG WKDW WKH % JLQJLYDOLV JHQRPH FDQ EH FORQHG DQG H[SUHVVHG LQ ( FROL 7KH FORQHG DQWLJHQV DUH SUHVHQWO\ EHLQJ LGHQWLILHG DQG IXUWKHU FKDUDFWHUL]HG IRU IXQFWLRQDO SURSHUWLHV 7KH FORQLQJ RI % JLQJLYDOLV JHQHV LV DQ DSSURDFK WKDW SURYLGHV QHZ WRROV IRU LQYHVWLJDWLRQV LQWR WKH SDWKRJHQHHLW\ RI % JLQJLYDOLV

PAGE 57

&+$37(5 7+5(( &+$5$&7(5,=$7,21 2) %$ &7(52,'(6 *,1*,9$/,6 $17,*(16 6<17+(6,=(' ,1 (6&+(5,&+,$ &2/, ,QWURGXFWLRQ %DFWHURLGHV JOQJLYDOLV SRVVHVVHV VHYHUDO SRWHQWLDO YLUXOHQFH IDFWRUV ZKLFK PD\ f SURPRWH LWV FRORQL]DWLRQ LQ WKH KRVW f UHVLVW KRVW GHIHQVHV DQG f FDXVH GHVWUXFWLRQ RI SHULRGRQWDO WLVVXHV 6ORWV DQG *HQFR f &RORQL]DWLRQ WKH LQLWLDO HYHQW LQ WKH HVWDEOLVKPHQW RI GLVHDVH UHTXLUHV WKH DGKHUHQFH RI EDFWHULD WR KRVW WLVVXHV *LEERQV DQG 9DQ +RXWH f WKHUHIRUH EDFWHULDO VXUIDFH FRPSRQHQWV ZKLFK PHGLDWH EDFWHULDO DGKHUHQFH DUH FRQVLGHUHG WR EH LPSRUWDQW YLUXOHQFH IDFWRUV ,Q WKH RUDO FDYLW\ EDFWHULD FDQ DWWDFK WR KRVW WLVVXHV DV ZHOO DV WR EDFWHULD LQ SUHIRUPHG SODTXH 6ORWV DQG *LEERQV f 7KH QDWXUH RI WKH ELQGLQJ VLWHV RQ WHHWK DQG RUDO WLVVXHV WR XnKLFK SHULRGRQWRSDWKLF EDFWHULD LQFOXGLQJ % JLQJLYDOLV DWWDFK KDV QRW EHHQ ZHOO HVWDEOLVKHG ,Q YLWUR % JLQJLYDOLV FDQ DWWDFK WR DQG DJJOXWLQDWH HU\WKURF\WHV 2NXGD DQG 7DND]RH 6ORWV DQG *LEERQV 6ORWV DQG *HQFR 2NXGD HW DO f FDQ DGKHUH LQ KLJK QXPEHUV WR KXPDQ EXFFDO HSLWKHOLDO FHOOV 6ORWV DQG *LEERQV 2NXGD HW DO f WR FUHYLFXODU HSLWKHOLDO FHOOV GHULYHG IURP SHULRGRQWDO SRFNHWV 6ORWV DQG *LEERQV f DQG WR VXUIDFHV RI *UDP SRVLWLYH EDFWHULD SUHVHQW LQ SODTXH 6ORWV DQG *LEERQV 6FKZDU] HW DO f ,Q DGGLWLRQ LW ZLOO DGKHUH WR XQWUHDWHG DQG VDOLYD WUHDWHG K\GUR[\DSDWLWH 6+$f EXW LQ FRPSDUDWLYHO\ ORZ QXPEHUV 6ORWV DQG *LEERQV f % JLQJLYDOLV KDV DOVR EHHQ UHSRUWHG WR ELQG WR

PAGE 58

+5 PDWUL[ D PDWHULDO VLPLODU WR WKH EDVHPHQW PHPEUDQH EDUULHU XQGHUO\LQJ FRQQHFWLYH WLVVXH /HRQJ HW DO f 5HFHQWO\ LW KDV EHHQ UHSRUWHG WKDW % JLQJLYDOLV FDQ ELQG WR ILEULQRJHQ DQG SRVVLEO\ FRORQL]H KRVW WLVVXH E\ DWWDFKLQJ WR ILEULQRJHQFRDWHG VXUIDFHV /DQW] HW DO f 6LQFH WKH FRPSRQHQWV LQYROYHG LQ % JLQJLYDOLV DGKHUHQFH LQ YLYR DUH DW SUHVHQW LOO GHILQHG WKH H[SUHVVLRQ RI DQ\ VWUXFWXUH GHWHFWHG E\ LQ YLWUR PHWKRGV WKXV QHHGV WR EH H[DPLQHG 7KHUHIRUH WKH DQWLJHQH[SUHVVLQJ FORQHV GHVFULEHG LQ &KDSWHU 7ZR ZHUH WHVWHG IRU WKH H[SUHVVLRQ RI DGKHVLQV IRU VDOLYDWUHDWHG K\GUR[\DSDWLWH 6+$ DGKHVLQf DQG HU\WKURF\WHV KHPDJJOXWLQLQf 7KLV FKDSWHU GHVFULEHV WKH DVVD\ IRU WKH 6+$ DGKHVLQ E\ WHVWLQJ IRU UHPRYDO RI 6+$ DGKHUHQFH LQKLELWLRQ E\ DQWL% JLQJLYDOLV DQWLVHUXP DQG WKH DVVD\ IRU KHPDJJOXWLQLQ E\ D GLUHFW KHPDJJOXWLQDWLRQ WHVW 7KH FORQHV ZKLFK ZHUH DEOH WR DJJOXWLQDWH HU\WKURF\WHV n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

PAGE 59

0DWHULDOV DQG 0HWKRGV %DFWHULDO 6WUDLQV DQG *URZWK &RQGLWLRQV %DFWHURLGHV JLQJLYDOLV ZDV FXOWXUHG LQ 7RGG+HZLWW EURWK DV GHVFULEHG LQ &KDSWHU 7ZR ( FROL WUDQVIRUPDQWV ZHUH FXOWXUHG LQ /% PHGLXP FRQWDLQLQJ PLFURJUDPV RI DPSLFLOOLQ SHU PO E\ SUHSDULQJ IROG GLOXWLRQV RI RYHUQLJKW FXOWXUHV IROORZHG E\ LQFXEDWLRQ IRU KRXUV 2 DW & ,37* ZDV DGGHG WR WKH FXOWXUHV ZKHQ XVHG DW D ILQDO FRQFHQWUDWLRQ RI P0 DQG WKH FXOWXUHV ZHUH LQFXEDWHG IRU DQ DGGLWLRQDO KRXUV $VVD\ IRU 5HPRYDO RI 6+$ $GKHUHQFH ,QKLELWLRQ E\ $QWL JLQJLYD-LV $QWLVHUXP $OLTXRWV RI DQWL% JLQJLYDOLV DQWLVHUXP ZHUH DGVRUEHG ZLWK HDFK DQWLJHQH[SUHVVLQJ FORQH DV ZHOO DV ( FROL -0 S8& f DV GHVFULEHG LQ &KDSWHU 7ZR 7KH WLWHU RI HDFK DGVRUEHG DQWLVHUXP ZDV WHVWHG DJDLQVW HH K FORQH DQG % JLQJLYDOLV ZKROH FHOO DQWLJHQ E\ (/,6$ DV GHVFULEHG DERYH :KROH SDUDIILQVWLPXODWHG KXPDQ VDOLYD ZDV FROOHFWHG DQG KHDWHG DW 2 & IRU PLQXWHV WR LQDFWLYDWH GHJUDGDWLYH HQ]\PHV ([WUDQHRXV GHEULV DQG FHOOV ZHUH UHPRYHG E\ FHQWULIXJDWLRQ DW USP IRU PLQXWHV DQG VRGLXP D]LGH ZnDV DGGHG WR D ILQDO FRQFHQWUDWLRQ RI b +\GUR[\DSDWLWH EHDGV +$f %'+ %LRFKHPLFDO /WG 3RROH (QJODQGf XUHUH WUHDWHG DV SUHYLRXVO\ GHVFULEHG &ODUN HW DO f %ULHIO\ PJ RI EHDGV ZHUH ZDVKHG DQG K\GUDWHG LQ GLVWLOOHG ZDWHU LQ PLFUROLWHU SODVWLF PLFURIXJH WXEHV IROORZHG E\ HTXLOLEUDWLRQ RYHUQLJKW ZLWK DGVRUSWLRQ EXIIHU 0 .& P0 .+32 S+ P0 &D&O

PAGE 60

DQG P0 0J&Kf 7KH EHDGV ZHUH LQFXEDWHG ZLWK PLFUROLWHUV RI VDOLYD IRU KRXUV DW r& DQG WKHQ ZDVKHG ZLWK VWHULOH DGVRUSWLRQ EXIIHU WR UHPRYH QRQDGVRUELQJ PDWHULDO &RQWURO WXEHV ZLWKRXW +$ ZHUH WUHDWHG LGHQWLFDOO\ % JLQJLYDOLV FHOOV ZHUH ODEHOHG E\ JURZWK WR ODWH ORJ SKDVH LQ PHGLXP VXSSOHPHQWHG ZLWK +f WK\PLGLQH P&LPOf 7KH FHOOV ZHUH SHOOHWHG ZDVKHG WZLFH LQ DGVRUSWLRQ EXIIHU DQG GLVSHUVHG ZLWK WKUHH VHFRQG SXOVHV PHGLXP SRZHUf ZLWK D PLFURXOWUDVRQLF FHOO GLVUXSWHU 7KH GLVSHUVHG FHOOV ZHUH PL[HG ZLWK HDFK DQWLVHUXP GLOXWLRQf DQG QRUPDO UDEELW VHUXP WR D ILQDO FRQFHQWUDWLRQ RI [ FHOOPO 7KH FHOODQWLVHUXP VXVSHQVLRQV PLFUROLWHUVf ZUHUH WKHQ DGGHG WR WKH 6+$ EHDGV LQ PLFURIXJH WXEHV DQG WKH WXEHV ZHUH URWDWHG LQ DQ DQDHURELF FKDPEHU IRU KRXU /DEHOHG FHOOV DORQH QR DQWLVHUDf ZHUH WUHDWHG LQ WKH VDPH PDQQHU WR GHWHUPLQH WKH QXPEHU RI FHOOV DGKHULQJ WR WKH 6+$ VXUIDFH $ FRQWURO WXEH FRQWDLQLQJ FHOOV EXW QR 6+$ ZUDV WHVWHG WR TXDQWLWDWH WKH DPRXQW RI FHOOV ERXQG WR WKH WXEHV UDWKHU WKDQ WR WKH 6+$ 2QH KXQGUHG PLFUROLWHUV RI DGVRUSWLRQ EXIIHU FRQWDLQLQJ XQDGKHUHG FHOOV ZUDV UHPRYHG DQG SODFHG LQ PLQLYLDOV FRQWDLQLQJ PO RI DTXHRXV VFLQWLOODWLRQ FRFNWDLO $PHUVKDP6HDUOH $UOLQJWRQ +HLJKWV ,/f DQG FRXQWHG ZLWK D 6FLQWLOODWLRQ &RXQWHU 0RGHO 3DUNDUG 7ULFDUEf 'HWHUPLQDWLRQ RI WKH QXPEHU RI FHOOV DGKHULQJ WR WKH 6+$ ZDV GRQH E\ VXEWUDFWLQJ WKH QXPEHU RI FHOOV QR RI FRXQWVf LQ VROXWLRQ IURP WKH WRWDO QXPEHU RI FHOOV QR RI FRXQWVf ZKLFK GLG QRW DGKHUH WR WKH WXEH

PAGE 61

'LUHFW +HPDJJOXWLQDWLRQ $VVD\ 7KH KHPDJJOXWLQDWLRQ DVVD\V ZHUH FDUULHG RXW LQ 9ERWWRP PLFURWLWHU SODWHV '\QDWHFK /DERUDWRULHV ,QF $OH[DQGULD 9LUJLQLDf (U\WKURF\WHV VKHHS RU KXPDQ JURXS f ZHUH ZDVKHG WLPHV ZLWK 3%6 0 SKRVSKDWH EXIIHUHG VDOLQHf S+ DQG UHVXVSHQGHG WR D ILQDO FRQFHQWUDWLRQ RI b YYf &HOOV RI % JLQJLYDOLV DQG DQWLJHQn H[SUHVVLQJ FORQHV ZHUH ZDVKHG WZLFH LQ 3%6 DQG UHVXVSHQGHG WR DQ RSWLFDO GHQVLW\ RI DQG UHVSHFWLYHO\ DW ULP 7KH FHOO VXVSHQVLRQV ZHUH GLOXWHG LQ D WZRIROG VHULHV ZLWK 3%6 DQG PO RI WKH VXVSHQVLRQV ZHUH DGGHG WR WKH ZHOOV ( FROL -0 S8& f ZKLFK ZDV SUHSDUHG LQ WKH VDPH PDQQHU DV WKH DQWLJHQH[SUHVVLQJ FORQHV ZDV LQFOXGHG DV D FRQWURO $Q HTXDO YROXPH POf RI ZDVKHG HU\WKURF\WHV ZDV DGGHG DQG PL[HG ZLWK WKH EDFWHULDO FHOOV 7KH SODWHV ZHUH VWRUHG IRU KRXUV DW r& DQG WKHQ H[DPLQHG IRU HYLGHQFH RI KHPDJJOXWLQDWLRQ DV IROORZV $JJOXWLQDWHG HU\WKURF\WHV ZLOO VHWWOH DV FOXPSV ZKLFK ZLOO EH GLVSHUVHG WKURXJKRXW WKH ERWWRP RI WKH ZHOOV UHVXOWLQJ LQ D SLQNLVKUHG FRDWLQJ RI HDFK ZHOO ,Q WKH DEVHQFH RI KHPDJJOXWLQDWLRQ WKH HU\WKURF\WHV ZLOO VHWWOH RQ WKH ERWWRP RI WKH ZHOO DV D FHQWUDO VPRRWK EULJKW UHG URXQG GLVN 7KH WLWHU ZDV H[SUHVVHG DV WKH UHFLSURFDO RI WKH KLJKHVW GLOXWLRQ VKRZLQJ SRVLWLYH DJJOXWLQDWLRQ +HPDJJOXWLQDWLRQ ,QKLELWLRQ $VVD\ 7KH KHPDJJOXWLQDWLRQ LQKLELWLRQ DVVD\ ZDV DOVR FDUULHG RXW LQ 9 ERWWRP PLFURWLWHU SODWHV % JLQJLYDOLV FHOO VXVSHQVLRQV LQ 3%6 XnHUH DGMXVWHG WR WKH RSWLFDO GHQVLW\ RI DW QP (DFK DQWLVHUXP H[DPLQHG IRU KHPDJJOXWLQDWLRQ LQKLELWLRQ DFWLYLW\ ZDV GLOXWHG WZRIROG

PAGE 62

LQ D VHULHV RI ZHOOV )LIW\ JO RI WKH EDFWHULDO VXVSHQVLRQ ZLWK WZLFH WKH PLQLPXP QXPEHU RI FHOOV ZKLFK SURGXFHG KHPDJJOXWLQDWLRQ ZDV WKHQ DGGHG WR HDFK ZHOO $IWHU LQFXEDWLRQ ZLWK JHQWOH VKDNLQJ DW URRP WHPSHUDWXUH IRU KRXU PO RI WKH ZnDVKHG HU\WKURF\WHV ZHUH DGGHG WR HDFK ZHOO DQG PL[HG 7KH SODWHV DUH OHIW IRU KRXUV DW & DQG UHDG IRU KHPDJJOXWLQDWLRQ DV GHVFULEHG DERYH IRU WKH KHPDJJOXWLQDWLRQ DVVD\ 7KH WLWHU ZDV H[SUHVVHG DV WKH UHFLSURFDO RI WKH KLJKHVW GLOXWLRQ VKRZLQJ KHPDJJOXWLQDWLRQ LQKLELWLRQ 3UHSDUDWLRQ RI $QWLVHUD WR +HPDJJOXWLQDEOH ( FROL ( FROL WUDQVIRUPDQWV ZKLFK ZHUH DEOH WR DJJOXWLQDWH HU\WKURF\WHV ZHUH JURZQ LQ /% EURWK FRQWDLQLQJ DPSLFLOOLQ DV GHVFULEHG DERYH 7ZnR UDEELWV ZnHUH LQMHFWHG ZGWK HDFK FORQH DV GHVFULEHG LQ &KDSWHU 7ZR 6HUD ZHUH H[KDXVWLYHO\ DGVRUEHG ZLWK ( FROL -0 S8& f DQG WHVWHG IRU DQWL% JLQJOYDOLV DFWLYLW\ E\ (/,6$ $GVRUSWLRQ RI $QWL&ORQH $QWLVHUXP $QWLFORQH DQWLVHUXP GLOXWHG ZDV VHSDUDWHO\ DGVRUEHG ZLWK % JLQJLYDOLV ( FROL -0 S8& f DQG FORQHV DQG :DVKHG VWDWLRQDU\ SKDVH FHOOV RI HDFK EDFWHULDO FXOWXUH ZHUH SUHSDUHG DV GHVFULEHG LQ &KDSWHU 7ZUR )RU HDFK DGVRUSWLRQ DQG EDFWHULDO FHOOV ZHUH PL[HG ZLWK XO RI VHUXP DQG WKH VXVSHQVLRQV ZHUH VWRUHG DW r& RYHUQLJKW 7KH VHUD ZHUH UHFRYHUHG E\ FHQWULIXJDWLRQ DW J IRU PLQXWHV (DFK DGVRUEHG DQWLVHUXP ZDV DVVD\HG E\ (/,6$ WR GHWHUPLQH WKH WLWHU WR % JLQJLYDOLV

PAGE 63

'1$ 3URFHGXUHV 5HVWULFWLRQ HQGRQXFOHDVH GLJHVWLRQV RI WKH UHFRPELQDQW SODVPLGV IURP FORQHV DQG ZHUH SHUIRUPHG DFFRUGLQJ WR PDQXIDFWXUHUnV GLUHFWLRQV 7KH VL]H RI '1$ LQVHUWV ZHUH HVWLPDWHG DQG 6RXWKHUQ EORW DQDO\VLV ZDV SHUIRUPHG DV GHVFULEHG LQ &KDSWHU 7ZR &ORQH '1$ ZDV GLJHVWHG ZLWK +LQG ,,, DQG WZR IUDJPHQWV RI % JLQJLYDOLV LQVHUWV ZHUH LVRODWHG IURP DJDURVH JHOV E\ WKH PHWKRG RI =KX HW DO f HPSOR\LQJ FHQWULIXJDO ILOWUDWLRQ RI '1$ IUDJPHQWV WKURXJK D 0LOOLSRUH PHPEUDQH LQVLGH D FRQLFDO WLS 7KH '1$ SUHSDUDWLRQV ZHUH H[WUDFWHG ZLWK SKHQRO FKORURIRUP SUHFLSLWDWHG ZLWK HWKDQRO DQG UHVXVSHQGHG LQ 7( S+ (DFK '1$ IUDJPHQW ZDV OLJDWHG WR +LQG ,,, GLJHVWHG S8& DQG WKH UHVXOWLQJ UHFRPELQDQW SODVPLGV ZHUH WUDQVIRUPHG LQWR FRPSHWHQW ( FROL -0 FHOOV DV GHVFULEHG LQ &KDSWHU 7ZR 5HFRPELQDQW SODVPLGV IURP WKHVH WUDQVIRUPDQWV ZHUH LVRODWHG E\ UDSLG SODVPLG '1$ LVRODWLRQ 6LOKDY\ HW DO f GLJHVWHG ZLWK DSSURSULDWH UHVWULFWLRQ HQGRQXFOHDVHV DQG DQDO\]HG E\ DJDURVH JHO HOHFWURSKRUHVLV 6RGLXP 'RGHF\O 6XOIDWH3RO\DFU\ODPLGH *HO (OHFWURSKRUHVLV 6'63$*(f DQG :HVWHUQ %ORW $QDO\VLV % JLQJLYDOLV FHOO O\VDWH DQG FHOOV RI ( FROL WUDQVIRUPDQW ZUHUH SUHSDUHG DQG DQDO\]HG E\ 6'63$*( DQG :HVWHUQ EORW WHFKQLTXHV DV GHVFULEHG LQ &KDSWHU 7ZR $QWLVHUD WR FORQHV DQG H[KDXVWLYHO\ DGVRUEHG ZLWK ( FROL -0 S8& f ZHUH XVHG DV SUREHV LQ WKH :HVWHUQ EORW &RQWURO DQWLVHUD LQFOXGHG DQWLFORQH DQWLVHUXP DOVR DGVRUEHG ZLWK % JLQJLYDOLV DW WKH UDWLR RI FHOOV SHU XO RI DQWLVHUXP DQG DQWLVHUXP WR ( FROL -0 KDUERULQJ S8& ZLWK (LKHQHOOD FRUURGHQV '1$ LQVHUW

PAGE 64

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

PAGE 65

7DEOH ,QKLELWLRQ RI DGKHUHQFH WR 6+$ E\ DGVRUEHG DQWL JLQJLYDOLV DQWLVHUD ,QKLELWRU DQG GLOXWLRQ b DGKHUHQFH b LQKLELWLRQ 1RQH 1RUPDO UDEELW VHUXP $QWLVHUXP XQDGVRUEHG $QWLVHUXP DGVRUEHG ZLWK ( FROL -0 S8& f &ORQH &ORQH &ORQH &ORQH &ORQH D 3HUFHQW DGKHUHQFH ZDV FDOFXODWHG IURP WKH IROORZLQJ IRUPXOD b DGKHUHQFH > &30 IURP WXEH ZLWKRXW 6+$ &30 IURP WXEH ZLWK 6+$f&30 IURP WXEH ZLWKRXW 6+$f@ [ E 3HUFHQW LQKLELWLRQ ZDV FDOFXODWHG IURP WKH IROORZLQJ IRUPXOD b LQKLELWLRQ &O b DGKHUHQFH LQ WKH SUHVHQFH RI DQWLERG\ b DGKHUHQFH LQ WKH DEVHQFH RI DQWLERG\f@ [

PAGE 66

FROL -0 S8& f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f GLG QRW )LJXUH f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f 7KH UHDUUDQJHPHQW PD\ KDYH UHVXOWHG IURP D GHOHWLRQ RU RWKHU UHDUUDQJHPHQW DW RQH +LQG ,,, HQG RI WKH LQVHUW DQG DQRWKHU +LQG ,,, HQG PD\ VWLOO EH LQWDFW ,Q RUGHU WR REWDLQ LQIRUPDWLRQ DV WR WKH QDWXUH RI WKH UHDUUDQJHPHQW RI FORQH DQG WKH UHODWLRQVKLS RI WKH WKUHH KHPDJJOXWLQDWLQJ FORQHV WR RQH DQRWKHU UHVWULFWLRQ PDSV RI WKHVH WKUHH FORQHV ZHUH JHQHUDWHG

PAGE 67

)LJXUH +HPDJJOXWLQDWLRQ RI VKHHS HU\WKURF\WHV %DFWHULDO VXVSHQVLRQV ZHUH GLOXWHG LQ IROG VHULDO GLOXWLRQV DQG PL[HG ZLWK DQ HTXDO YROXPH RI b 99f HU\WKURF\WHV DV GHVFULEHG LQ WKH PHWKRGV 5RZ LV WKH XQGLOXWHG EDFWHULDO VXVSHQVLRQV $f % JLQJLYDOLV %f ( RROL -0 S8& f &f FORQH 'f &ORQH (f &ORQH )f &ORQH *f &ORQH

PAGE 69

7KH UHFRPELQDQW SODVPLGV RI FORQHV DQG ZHUH UHVWULFWHG ZLWK VHYHUDO UHVWULFWLRQ HQGRQXFOHDVHV DQG DQDO\]HG LQ b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f &ORQH '1$ UHVWULFWHG ZLWK +LQG ,,, UHVXOWHG LQ IUDJPHQWV RI S8& DQG SLHFHV RI LQVHUW RI DSSUR[LPDWHO\ DQG ES )LJXUH SDQHO $ ODQH f )UDJPHQW EDQGV RI S8& DQG LQVHUWV RI DSSUR[LPDWHO\ DQG ES ZHUH JHQHUDWHG IURP GLJHVWLRQ RI FORQH '1$ ZLWK +LQG ,,, DQG %DP +, )LJXUH SDQHO $ ODQH f &ORQH '1$

PAGE 70

)LJXUH $JDURVH JHO HOHFWURSKRUHVLV RI UHVWULFWLRQ GLJHVWV RI WKH UHFRPELQDQW SODVPLG IURP FORQH /DQHV '1$ PDUNHU+LQG ,,, GLJHVW RI ODPEGD '1$ S8& GLJHVWHG ZLWK +LQG ,,, S8& GLJHVWHG ZLWK 3YX ,, DQG UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, 3YX ,, 3YX ,, DQG +LQG ,,, (FR 5, (FR 5, DQG +LQG ,,, 6PD 6PD DQG +LQG ,,, 6PD DQG (FR 5, (FR 5, DQG 3YX ,, 1DU (FR 5, DQG 1DU %DP +, +LQG ,,, DQG %DP +, 6PD DQG %DP +, &D &D DQG +LQG ,,, (FR 5, DQG &D ,

PAGE 71

L A rrVSS ‘ n f r \ \0We X L r fL Z : mf 9 A m0 m0 rr r} 0 fn‘ f f ,1, WIFD 0O X f f B r‘ } f A n } ff L 0 8 PX OL 0 A ‘]6In MIU ‘ 0 r f r f Uf !n nY f f r r fr f }:W nfn‘f :Y f "Y9 Y? f IH f &7! 32

PAGE 72

)LJXUH $JDURVH JHO HOHFWURSKRUHVLV RI UHVWULFWLRQ GLJHVWV RI WKH UHFRPELQDQW SODVPLG IURP FORQH /DQHV '1$ PDUNHU+LQG ,,, GLJHVW RI ODPEGD '1$ S8& GLJHVWHG ZLWK +LQG ,,, DQG UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, %DP +, +LQG ,,, DQG %DP +, 6DO +LQG ,,, DQG 6DO +LQG ,,, DQG (FR 59 6WX +LQG ,,, DQG 6WX 6WX DQG 6DO 6WX DQG (FR 59 6WX DQG $VS +LQG ,,, DQG $VS %DP +, DQG $VS %DP +, DQG 6WX (FR 5, +LQG ,,, DQG (FR 5,

PAGE 73

&7O 3r

PAGE 74

)LJXUH $JDURVH JHO HOHFWURSKRUHVLV RI UHVWULFWLRQ GLJHVWV RI UHFRPELQDQW SODVPLGV IURP FORQHV DQG /DQHV '0$ PDUNHU+LQG ,,, GLJHVW RI ODPEGD '1$ S8& GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLGV IURP FORQHV DQG LQ DGMDFHQW ODQHV GLJHVWHG ZLWK DQG +LQG ,,, DQG +LQG ,,, DQG %DP +, DQG +LQG ,,, DQG $VS DQG +LQG ,,, DQG 6WX DQG 6DO DQG +LQG ,,, DQG (FR 5,

PAGE 75

&7! &7L

PAGE 76

)LJXUH 5(VWULFWLRQ PDS RI WKH UHFRPELQDQW SODVPLG IURP FORQH 7KH KHDY\ OLQH UHSUHVHQWV % JLQJLYDOLV '1$ LQVHUW

PAGE 77

3YX ,, +LQG ,,, %DP +, +LQG +, %DP +, &D 6PD 1DU &D 6PD 1DU (FR5, FR I2 E R R R R R R FU ;f FUA

PAGE 78

)LJXUH 5HVWULFWLRQ PDS RI WKH UHFRPELQDQW SODVPLG IURP FORQH 7KH KHDY\ OLQH UHSUHVHQWV % JLQJLYDOLV '0$ LQVHUW

PAGE 79

+LQG ,,, (FR 59 6DO 6WX +LQG ,,, %DP +, $VS 6WX (FR 5O ‘ +LQG ,,, f§ 3VW f§ 6DO ‘f§%DP +, f§ 6PD f(FR 5O R R R Lf§ R FU fR R

PAGE 80

)LJXUH 5HVWULFWLRQ PDS RI WKH UHFRPELQDQW SODVPLG IURP FORQH 7KH KHDY\ OLQH UHSUHVHQWV % DLQJLYDOLV '1$ LQVHUW

PAGE 81

+LQG ,,, 3VW 6DO %DP +, 6PD (FR 5O FU ‘R R

PAGE 82

)LJXUH 6FKHPDWLF GLDJUDP RI UHVWULFWLRQ HQ]\PH UHFRJQLWLRQ VLWHV RI UHFRPELQDQW SODVPLGV IURP FORQHV DQG 7KH VROLG OLQHV UHSUHVHQW % JLQJLYDOLV '1$ LQVHUWV 7KH KDWFKHG ER[HV UHSUHVHQW S8& UHJLRQV

PAGE 83

12 =<$f§L Lf§Q <$n +LQDf nm LD D rp f§ &D 6PD N 0DULf§ m‘ + r! (FR )8 +LQG ,,, 3VW 6DO %DP + 6PD (FR 5 +LQG ,,, 3VW 6DO %DP + 6PD (FR 5 2 Of R ] P UR 2 Ua 2 ] P FQ 8

PAGE 84

)LJXUH 6RXWKHUQ EORW DQDO\VLV RI WKH KHPDJJOXWLQDWLQJ ( RROL $f $JDURVH JHO bf VKRZLQJ UHVWULFWLRQ GLJHVWV RI WKH '1$ /DQHV S8& GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, (FR 5, DQG 6PD UHFRPn ELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, DQG %DP +, UHFRPELQDQW SODVPLG IURP FORQH GLJHVWHG ZLWK +LQG ,,, DQG %DP +, %f $XWRUDGLRJUDSK RI '1$ LQ SDQHO $ DIWHU 6RXWKHUQ WUDQVIHU DQG K\EULGL]DWLRQ ZLWK SBLDEHOHG UHFRPELQDQW '1$ IURP FORQH

PAGE 86

UHVWULFWHG ZLWK +LQG ,,, DORQH DQG +LQG ,,, WRJHWKHU ZnLWK %DP +, UHVXOWHG LQ S8& DQG DQ LQVHUW RI ES )LJXUH SDQHO $ ODQH f DQG S8& LQVHUW RI DQG ES )LJXUH SDQHO $ ODQH f UHVSHFWLYHO\ +\EULGL]DWLRQ RI WKHVH WUDQVIHUUHG UHVWULFWHG '1$V GHPRQVWUDWHG WKDW WKH FORQH SUREH K\EULGL]HG WR S8& DQG WKH FRPPRQ LQVHUW RI FORQHV DQG EXW QRW WR WKH LQVHUW RI FORQH )LJXUH SDQHO %f 6XEFORQLQJ RI &ORQH IRU $XWRDJJOXWLQDWLRQ DQG +HPDJJOXWLQDWLRQ &ORQH ZDV IRXQG WR DJJOXWLQDWH HU\WKURF\WHV DQG DXWRDJJOXWLQDWH ZKLOH FORQH ZDV RQO\ DEOH WR DJJOXWLQDWH HU\WKURF\WHV &ORQH KDV DQ LQVHUW RI ES LQ DGGLWLRQ WR WKH FRPPRQ LQVHUW RI ES RI FORQH 7KLV GDWD VXJJHVWHG WKDW WKH ES LQVHUW PLJKW HQFRGH IRU WKH DXWRDJJOXWLQDWLQJ DFWLYLW\ DQG WKH ES IUDJPHQW IRU WKH KHPDJJOXWLQDWLQJ DFWLYLW\ RI FORQH 7KH UHFRPELQDQW SODVPLG RI FORQH ZDV WKXV GLJHVWHG ZLWK +LQG ,,, WR JHQHUDWH S8& DQG LQVHUWV RI DQG ES (DFK LQVHUW EDQG ZDV LVRODWHG IURP WKH DJDURVH JHO DQG OLJDWHG WR +LQG ,,, FXW S8& DQG WUDQVIRUPHG LQWR ( FROL -0 7KH SODVPLGV ZnHUH LVRODWHG IURP WKHVH WUDQVIRUPDQWV DQG GLJHVWHG ZLWK UHVWULFWLRQ HQGRQXFOHDVHV 6XEFORQHV ZLWK GLIIHUHQW RULHQWDWLRQV RI WKH LQVHUW ZHUH REWDLQHG 6XEFORQHV RI ES LQVHUWV ZnHUH GHVLJQDWHG FORQH DQG DQG WKH VXEFORQHV RI ES LQVHUWV FORQH DQG 5HFRPELQDQW SODVPLGV RI FORQHV DQG GLJHVWHG ZGWK +LQG ,,, GLG UHVXOW LQ S8& DQG WKH ES LQVHUWV )LJXUH ODQHV DQG f DQG GLIIHUHQW SDWWHUQV RI UHVWULFWHG '1$V ZHUH VHHQ ZKHQ GLJHVWHG ZLWK 6DO )LJXUH ODQHV DQG f +LQG ,OO UHVWULFWHG UHFRPELQDQW SODVPLGV RI FORQHV DQG UHYHDOHG S8&

PAGE 87

DQG LQVHUWV RI ES )LJXUH LDQHV DQG f ZKLOH (FR 5, UHVWULFWHG UHFRPELQDQW SODVPLGV VKRZHG GLIIHUHQW SDWWHUQV )LJXUH ODQHV DQG f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f ,Q DQ DWWHPSW WR GHWHFW DQWLJHQ H[SUHVVLRQ RI '1$ LQVHUWV LQ FORQHV DQG DQG WR DFKLHYH H[SUHVVLRQ RI D PRUH VWDEOH SURGXFW IURP FORQH WKH UHFRPELQDQW SODVPLGV IURP WKHVH FORQHV ZHUH WUDQVIRUPHG LQWR ( FROL /& OKWS5$P7Vf ORQ5 7Vf ODFL$Pf WUS^$Pf SKR$Pf USV/ VXS&L7Vf PDO$Pf WV[7QO2@ NLQGO\ SURYLGHG E\ $ / *ROGEHUJ 7KLV EDFWHULDO VWUDLQ KDV PXWDWLRQV LQ WKH KWS5 DQG ,RQ JHQHV WKH SURGXFWV RI ZKLFK DUH LQYROYHG LQ LQWUDFHOOXODU SURWHLQ GHJUDGDWLRQ +RZHYHU WKLV DWWHPSW ZDV QRW VXFFHVVIXO VLQFH WKH H[SUHVVHG DQWLJHQ RI FORQH ZDV VWLOO GHJUDGHG DQG DQWLJHQ H[SUHVVLRQ RI FORQHV DQG ZDV QRW GHWHFWHG ,GHQWLILFDWLRQ RI 1DWLYH % JLQJLYDOLV $QWLJHQV ,Q RUGHU WR GHWHUPLQH WKH QDWLYH % JLQJLYDOLV DQWLJHQV ZKLFK FORQH H[SUHVVHG DQWLVHUD DJDLQVW FORQH ZHUH PDGH LQ UDEELWV IRU

PAGE 88

)LJXUH $JDURVH JHO HOHFWURSKRUHVLV RI UHFRPELQDQW SODVPLGV IURP FORQHV DQG /DQHV '1$ PDUNHU+LQG ,,, GLJHVW RI ODPEGD '1$ UHFRPELQDQW SODVPLGV IURP FORQHV DQG GLJHVWHG ZLWK +LQG ,,, DQG UHFRPELQDQW SODVPLGV IURP FORQHV DQG GLJHVWHG ZLWK 6DO DQG UHFRPELQDQW SODVPLGV IURP FORQHV DQG GLJHVWHG ZLWK (FR 5,

PAGE 90

XVH DV D SUREH LQ :HVWHUQ EORW DQDO\VLV 3RROHG DQWLFORQH DQWLVHUXP KDG D WLWHU RI DJDLQVW % JLQJLYDOLV ZKROH FHOO DQWLJHQ 7KLV DQWLVHUXP ZDV DGVRUEHG H[KDXVWLYHO\ ZLWK ( FROL -0 S8& f XQWLO WKH DQWL)/ FROL WLWHU ZDV UHGXFHG IURP WR LQ WKH ( FROL ZKROH FHOO (/,6$ 7KH DGVRUEHG DQWLVHUXP GLOXWHG WR ZDV XVHG DV D SUREH WR GHWHFW DQWLJHQV VHSDUDWHG LQ D b 6'6 SRO\DFU\ODPLGH JHO DQG WUDQVIHUUHG WR D QLWURFHOOXORVH VKHHW $V FDQ EH VHHQ LQ )LJXUH WKLV DQWLVHUXP UHDFWHG ZLWK PDMRU EDQGV RI DSSUR[LPDWHO\ 0:V DQG DQG EDQGV RI 0:V DQG LQ % JLQJLYDOLV FHOO O\VDWH DQWLJHQ DQG WKH SURWHLQ EDQG RI H[SUHVVHG DQWLJHQ LQ FORQH 1RUPDO UDEELW VHUXP UHDFWHG WR D FRPPRQ PROHFXODU ZHLJKW EDQG RI DOO WKH FORQHV DQG ( FROL -0 S8& f ,Q RUGHU WR SURYH WKDW WKH % JLQJLYDOLV UHDFWLYH SRO\SHSWLGHV DUH H[FOXVLYHO\ % JLQJLYDOLV SURWHLQV WKH QDWLYH % JLQJLYDOLV DQWLJHQV ZHUH UHDFWHG WR ( FROL DGVRUEHG DQWLFORQH DQWLVHUXP % JLQJLYDOLV FHOO O\VDWH DQWLJHQ DQG FORQH ZKROH FHOO DQWLJHQ ZHUH DJDLQ VHSDUDWHG LQ b 6'6 SRO\DFU\ODPLGH JHO 8SRQ WUDQVIHU WR D QLWURFHOOXORVH VKHHW HDFK ZDV UHDFWHG ZLWK f ( FROL DGVRUEHG DQWLFORQH DQWLVHUXP f % JLQJLYDOLV DGVRUEHG DQWLFORQH DQWLVHUXP DQG f DQWLVHUD WR ( FROL -0 KDUERULQJ S8& ZLWK DQ (LNHQHOOD FRUURGHQV '1$ LQVHUW $V FDQ EH VHHQ LQ )LJXUH ( FROL DGVRUEHG DQWLFORQH UHDFWHG WR % JLQJLYDOLV FHOO O\VDWH DW PDMRU EDQGV RI 0:V DQG EDQGV RI 0:V DQG DQG IDLQW EDQGV RI KLJKHU PROHFXODU ZHLJKW RI DSSUR[LPDWHO\ DQG GDOWRQV 7KLV DGVRUEHG DQWLVHUXP DOVR UHDFWHG WR D 0: EDQG RI H[SUHVVHG DQWLJHQ LQ FORQH % JLQJLYDOLV DGVRUEHG DQWLFORQH DQG DQWL ( FROL -0 KDUERULQJ S8& ZLWK (LNHQHOOD '1$ LQVHUW DQWLVHUD GLG

PAGE 91

)LJXUH :HVWHUQ EORW DQDO\VLV RI QDWLYH % JLQJLYDOLV DQWLJHQV H[SUHVVHG E\ FORQH /DQHV % JLQJLYDOLV FHOO O\VDWH SJf WR ZKROH FHOO VDPSOHV RI FORQHV DQG ( DROL -0 S8& f DV GHVFULEHG LQ WKH PHWKRGV $f 7KH EORW ZDV SUREHG ZLWK ( FROLDGVRUEHG DQWLFORQH DQWLVHUXP %f 7KH EORW ZDV SUREHG ZLWK QRUPDO UDEELW VHUXP

PAGE 93

)LJXUH :HVWHUQ EORW DQDO\VLV RI QDWLYH % JLQJLYDOLV DQWLJHQV H[SUHVVHG E\ FORQH /DQHV % JLQJLYDOLV FHOO O\VDWH XJf ZKROH FHOO VDPSOH RI FORQH $f 7KH EORW ZDV SUREHG ZLWK ( FROL DGVRUEHG DQWLFORQH DQWLVHUXP %f 7KH EORW ZDV SUREHG ZLWK % JLQJLYDOLV DGVRUEHG DQWLFORQH DQWL VHUXP &f 7KH EORW ZDV SUREHG ZLWK DQWLVHUXP DJDLQVW ( FROL -0 KDUERULQJ S8& ZLWK DQ (LNHQHOOD '1$ LQVHUW

PAGE 94

$ . . . % & frmr"rr ILUrn

PAGE 95

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f 7KLV DQWLVHUXP DOVR UHDFWHG WR D FRPPRQ EDQG RI DSSUR[LPDWHO\ GDOWRQV RI ( FROL DQWLJHQ LQ HDFK FORQH DQG ( FROL -0 S8& f 1RUPDO UDEELW VHUXP GLG QRW UHDFW WR DQ\ % JLQJLYDOLV DQWLJHQV )LJXUH f ,Q RUGHU WR GHWHUPLQH LI WKH DQWLFORQHV DQG DQWLVHUD ZHUH UHDFWLQJ ZLWK WKH VDPH % JLQJLYDOLV SRO\SHSWLGHV RU ZLWK GLIIHUHQW SHSWLGHV RI VLPLODU PLJUDWLRQ UDWHV IRXU VDPSOHV RI % JLQJLYDOLV FHOO O\VDWH DQWLJHQV ZHUH VHSDUDWHG LQ D b 6'6 SRO\DFU\ODPLGH JHO WUDQVIHUUHG WR QLWURFHOOXORVH SDSHU DQG UHDFWHG ZLWK DQWLFORQH DQWLVHUXP GLOXWHG DQWLFORQH DQWLVHUXP GLOXWHG DQWLFORQH DQWLVHUXP GLOXWHG DQG D PL[WXUH RI DQWLFORQHV DULG DQWLVHUD DW ILQDO FRQFHQWUDWLRQV RI DQG UHVSHFWLYHO\ $Q\ GLIIHUHQFHV LQ WKH SDWWHUQ RI UHDFWLRQ ZHUH XQGLVFHUQDEOH LQ WKHVH EORWV )LJXUH f

PAGE 96

)LJXUH :HVWHUQ EORW DQDO\VLV RI QDWLYH % JLQJLYDOLV DQWLJHQ H[SUHVVHG E\ FORQH /DQHV % JLQJLYDOLV FHOO O\VDWH XJf WR ZKROH FHOO VDPSOHV RI FORQHV DQG ( FROL -0 S8& f DV GHVFULEHG LQ WKH PHWKRGV $f 7KH EORW ZDV SUREHG ZLWK ( FROLDGVRUEHG DQWLFORQH DQWLVHUXP %f 7KH EORW ZDV SUREHG ZLWK QRUPDO UDEELW VHUXP

PAGE 97

$ . . . Y0} f 5 ‘!r % RR RR

PAGE 98

)LJXUH :HVWHUQ EORW DQDO\VLV RI QDWLYH % JLQJLYDOLV DQWLJHQV H[SUHVVHG E\ FORQHV DQG )RUW\ XJ RI % JLQJLYDOLV FHOO O\VDWH ZDV VHSDUDWHG LQ D b 6'6 SRO\DFU\ODPLGH JHO WUDQVIHUUHG WR D QLWURFHOOXORVH VKHHW DQG SUREHG ZLWK f ( FROL DGVRUEHG DQWLFORQH DQWLVHUXP GLOXWHG f ( FROL DGVRUEHG DQWLFORQH DQWLVHUXP GLOXWHG f ( FROL DGVRUEHG DQWLFORQH DQWLVHUXP GLOXWHG DQG f D PL[WXUH RI WKH DERYH DQWLVHUD

PAGE 99

BB @ . . .

PAGE 100

'HWHUPLQDWLRQ RI 7KH 5HODWLRQVKLS EHWZHHQ WKH &ORQHV DQG B H[SUHVVHG DQWLJHQV $OWKRXJK DQWLVHUD DJDLQVW FORQHV DQG UHDFWHG WR % JLQJLYDOLV FHOO O\VDWH DW PDMRU EDQGV RI DQG 0:V )LJXUH ODQH RI SDQHO $ % DQG &f ( FROL DGVRUEHG DQWLFORQH DQWLVHUXP DOVR UHDFWHG WR WKH SURWHLQ EDQG V\QWKHVL]HG LQ FORQH )LJXUH SDQHO $ ODQH f +RZHYHU ( FROL DGVRUEHG DQWLFORQH DQG DQWLFORQH DQWLVHUD GLG QRW UHDFW WR WKLV H[SUHVVHG DQWLJHQ EDQG RI FORQH )LJXUH ODQH RI SDQHO % DQG &f 7R IXUWKHU GHILQH WKH UHODWLRQVKLS RI WKH HSLWRSHV RI WKH H[SUHVVHG DQWLJHQ LQ FORQH IURP WKDW RI FORQHV DQG DGVRUSWLRQ RI DQWLFORQH DQWLVHUXP ZLWK VHYHUDO DQWLJHQV ZDV SHUIRUPHG DQG HDFK DGVRUEHG DQWLFORQH DQWLVHUXP ZDV WHVWHG IRU LWV WLWHU WR % JLQJLYDOLV ZKROH FHOO DQWLJHQ E\ (/,6$ 7KHVH UHVXOWV ZHUH DV VKRZQ LQ )LJXUH 7KH DQWLERG\ WLWHU WR % JLQJLYDOLV RI DQWLFORQH DQWLVHUXP ZDV UHPRYHG LQ D GRVH UHVSRQVH PDQQHU E\ DGVRUSWLRQ ZLWK % JLQJLYDOLV DQG FORQH FHOOV $GVRUSWLRQ ZLWK ( FROL -0 S8& f FORQH RU FORQH GLG QRW UHGXFH WKH DQWLERG\ WLWHU WR % JLQJLYDOLV RI DQWLFORQH DQWLVHUXP +HPDJJOXWLQDWLRQ ,QKLELWLRQ 7KH DELOLW\ RI DQWLVHUD WR % JLQJLYDOLV DQG KHPDJJOXWLQDEOH ( FROL WR LQKLELW WKH KHPDJJOXWLQDWLQJ DFWLYLW\ RI % JLQJLYDOLV ZDV GHWHUPLQHG DQG LV VXPPDUL]HG LQ 7DEOH $LO DQWLVHUD LQKLELWHG % JLQJLYDOLV KHPDJJOXWLQDWLRQ DW WLWHUV WR WLPHV WKDW RI QRUPDO UDEELW VHUD

PAGE 101

)LJXUH 'HWHFWLRQ RI % JLQJLYDOLV DQWLJHQV V\QWKHVL]HG E\ FORQHV DQG DV GHWHUPLQHG E\ :HVWHUQ EORW DQDO\VLV /DQHV % JLQJLYDOLV FHOO O\VDWH XJf ZKROH FHOO VDPSOH RI FORQH $f 7KH EORW ZDV SUREHG ZLWK ( FROL DGVRUEHG DQWLFORQH GLOXWHG %f 7KH EORW ZDV SUREHG ZLWK ( FROL DGVRUEHG DQWLFORQH GLOXWHG &f 7KH EORW ZDV SUREHG ZLWK ( FROL DGVRUEHG DQWLFORQH GLOXWHG

PAGE 102

. . . .

PAGE 103

)LJXUH (/,6$ RI DQWLFORQH DQWLVHUXP DGVRUEHG ZLWK YDULRXV QXPEHUV RI FHOOV RI % JLQJLYDOLV R f ( DROL -0 KDUERULQJ S8& f f FORQH Df FORQH Df DQG FORQH Df 7KH DGVRUEHG DQWLVHUD ZHUH DGGHG WR % JLQJLYDOLVFRDWHG PLFURWLWHU SODWHV DQG WKH DVVD\ ZDV SHUIRUPHG DV GHVFULEHG LQ WKH PHWKRGV

PAGE 104

2' QP

PAGE 105

7DEOH ,QKLELWLRQ RI KHPDJJOXWLQDWLQJ DFWLYLW\ RI % JLQJLYDOLV E\ DQWLKHPDJJOXWLQDWLQJ ( FROL DQWLVHUD $QWLVHUXP +HPDJJOXWLQDWLRQ LQKLELWLRQ WLWHU $QWL% JLQJLYDOLV XQDGVRUEHG DGVRUEHG ZLWK ( FROL -0 S8& f 1RUPDO UDEELW VHUXP $QWLFORQH 3UHLPPXQH $QWLFORQH 3UHLPPXQH $QWLFORQH 3UHLPPXQH 1RUPDO UDEELW VHUXP DQG SUHLPPXQH VHUD WLWHUV DUH IURP HDFK SDUWLFXODU JURXS RI UDEELWV

PAGE 106

7KH DELOLW\ RI HDFK KHPDJJOXWLQDWLQJ FORQH WR UHPRYH WKH KHPDJJOXn WLQDWLRQ LQKLELWLRQ DFWLYLW\ RI DQWL' JLQJLYDLLV DQWLVHUXP ZDV WHVWHG DQG WKH UHVXOWV DUH VKRZQ LQ 7DEOH (DFK FORQH SDUWLDOO\ UHPRYHG KHPDJJOXWLQDWLRQ LQKLELWLRQ DFWLYLW\ RI DQWL% JLQJLYDLLV DQWLVHUXP &ORQHV DQG GHFUHDVHG WKH KHPDJJOXWLQDWLRQ LQKLELWLRQ WLWHU RI % JLQJLYDLLV DQWLVHUXP WR IROG $GVRUSWLRQ RI WKH DQWLVHUD ZLWK FORQH FHOOV UHGXFHG WKH WLWHU WR IROG DQG D PL[WXUH RI WKHVH FORQHV UHGXFHG WKH WLWHU IROG % JLQJLYDLLV LWVHOI UHGXFHG WKH WLWHU IROG DQG ( FROL -0 S8& f KDG QR HIIHFW RQ WKH LQKLELWLRQ DFWLYLW\ RI WKH DQWLVHUXP 'LVFXVVLRQ :KHQ DQWLJHQH[SUHVVLQJ FORQHV ZHUH VXUYH\HG IRU IXQFWLRQDO DFWLYLWLHV FORQHV DQG ZHUH DEOH WR DJJOXWLQDWH HU\WKURF\WHV ZKHUHDV ( FROL -0 S8& f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

PAGE 107

7DEOH ,QKLELWLRQ RI KHPDJJOXWLQDWLQJ DFWLYLW\ RI % JLQJLYDOLV E\ DGVRUEHG DQWL JLQJLYDOLV DQWLVHUXP $QWLVHUXP +HPDJJOXWLQDWLRQ LQKLELWLRQ WLWHU $QWL% JLQJLYDOLV XQDGVRUEHG DGVRUEHG ZLWK ( FROL -0 S8& f DGVRUEHG ZLWK % JLQJLYDOLV DGVRUEHG ZLWK FORQH DGVRUEHG ZLWK FORQH DGVRUEHG ZLWK FORQH DGVRUEHG ZLWK FORQHV DQG

PAGE 108

DQG FRXOG QRW EH GHWHFWHG E\ :HVWHUQ EORW DQDO\VLV 2WKHU WHFKQLTXHV VXFK DV LQ YLWUR WUDQVFULSWLRQWUDQVODWLRQ DQG LPUDXQRSUHFLSLWDWLRQ PLJKW EH DSSURSULDWH WR GHWHFW WKH H[SUHVVHG DQWLJHQV 7R GDWH WKH KHPDJJOXWLQLQ RI % JLQJLYDOLV VWUDLQ KDV EHHQ SDUWLDOO\ SXULILHG LQ GLIIHUHQW ODERUDWRULHV 7KH SDUWLDOO\ SXULILHG KHPDJJOXWLQLQ LVRODWHG E\ ,QRVKLWD HW DO f VHSDUDWHG E\ 6'63$*( GHPRQVWUDWHG PDMRU SURWHLQ EDQGV RI 0:V . DQG DV ZHOO DV EDQGV DW DQG JUHDWHU WKDQ 2NXGD HW DO f DOVR SXULILHG D KHPDJJOXWLQLQ RI ZKLFK WKH 6'63$*( SDWWHUQ FRQWDLQHG D PDMRU SURWHLQ EDQG RI LQ DGGLWLRQ WR VHYHUDO SURWHLQ EDQGV LQ OHVVHU DPRXQWV $ SUHYLRXV UHSRUW E\ 1DLWR HW DO f GHPRQVWUDWHG WKDW PRQRFORQDO DQWLERG\ GLUHFWHG DJDLQVW % JLQJLYDOLV KHPDJJOXWLQLQ EXW QRW /36 RU FDSVXOHf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

PAGE 109

VWURQJO\ VXJJHVWV WKDW WKH % JLQJLYDOLV KHPDJJOXWLQLQ LV H[SUHVVHG LQ FORQH 7KH '1$ LQVHUW RI FORQH PLJKW HQFRGH IRU DOO IRXU RI WKH SRO\SHSWLGHV RI . DQG RU RQO\ D SRUWLRQ RI VRPH RI WKHVH SRO\SHSWLGHV RI % JLQJLYDOLV ( FROL -0 KDV D QRQVHQVH VXSSUHVVRU W51$ LQ ZKLFK W51$ LV DEOH WR VXSSUHVV WKH WHUPLQDWLRQ FRGRQ 8$*f DW WKH HQG RI WKH JHQH WKDW XVHV WKLV FRGRQ 7KHUHIRUH WKH P51$ PD\ EH UHDG WKURXJK UHVXOWLQJ LQ WKH V\QWKHVLV RI WKH LQWDFW SURWHLQ EDQG RI ZKLFK PLJKW DOVR FRQWDLQ D SHSWLGH RI SDUW RI WKH SJDODFWRVLGDVH SHSWLGH 7KH SRO\SHSWLGH PLJKW EH SDUW RI WKH SHSWLGH VLQFH LW KDV EHHQ VKRZQ WKDW PRQRFORQDO DQWLERG\ UHDFWV WR EDQGV RI VLPLODU PROHFXODU ZHLJKWV 1DLWR HW DO f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

PAGE 110

DV KHPDJJOXWLQLQV 7ZR SRVVLELOLWLHV WKDW PD\ H[SODLQ WKLV SKHQRPHQRQ DUH f GLIIHUHQW %DFWHURLGHV FRPSRQHQWV PLJUDWHG LQ 6'63$*( WKH VDPH GLVWDQFH 7KLV KRZHYHU DSSHDUV XQOLNHO\ 7R UHVROYH WKHVH EDQGV D WHFKQLTXH RI WZRGLPHQVLRQDO JHO HOHFWURSKRUHVLV PXVW EH SHUIRUPHG f HDFK FORQHG LQVHUW HQFRGHV D GLIIHUHQW SRUWLRQ RI WKH VDPH SRO\SHSWLGH RI % JLQJLYDOLV DQG HDFK SRUWLRQ ZLWK GLIIHUHQW DQWLJHQLF HSLWRSHV FDQ IXQFWLRQ DV D KHPDJJOXWLQDWLRQ ,Q WKH ODWWHU FDVH WKH FORQH LQVHUW FRQWDLQV D %DFWHURLGHV SURPRWHU EXW WKH FORQH LQVHUW GRHV QRW 7KH DQG SRO\SHSWLGHV PLJKW EH JHQHUDWHG IURP D VLQJOH JHQH E\ VWDUWLQJ RU WHUPLQDWLQJf H[SUHVVLRQ DW GLIIHUHQW SRLQWV 7KH FORQH LQVHUW RU SDUW RI WKH LQVHUW ZRXOG FRGH IRU WKH 1WHUPLQXV RI WKH SRO\SHSWLGH DQG HQG VRPH ZKHUH DW WKH 1WHUPLQXV RI WKH SRO\SHSWLGH 7KH FORQH LQVHUW PD\ FRGH IRU WKH VDPH &WHUPLQXV RI WKH DQG SRO\SHSWLGHV DQG PLJKW FRQWDLQ WKH VWUXFWXUDO JHQH IRU RWKHU SHSWLGH DQG .f WR Xn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

PAGE 111

IXQFWLRQDO HSLWRSHV RI WKH VDPH SRO\SHSWLGH RU WKH FDVH RI GLIIHUHQW KHPDJJOXWLQLQ FRPSRQHQWV

PAGE 112

&+$37(5 )285 &21&/86,21 7KH KHPDJJOXWLQDWLQJ DFWLYLW\ RI % JLQJLYDOLV KDV EHHQ VWXGLHG DV D SDUDPHWHU WKDW DIIHFWV WKH DGKHUHQFH RI WKLV RUJDQLVP LQ WKH SHULRGRQWDO SRFNHW VLQFH % JLQJLYDOLV SRVVHVVHV ILPEULDH 2NXGD DQG 7DND]RH 6ORWV DQG *LEERQV f DQG PDQ\ ILPEULDH RI RWKHU EDFWHULDO VSHFLHV KDYH KHPDJJOXWLQDWLQJ DFWLYLW\ DV ZHOO DV WKH DELOLW\ WR DGKHUH WR D YDULHW\ RI KRVW FHOOV 3HDUFH DQG %XFKDQDQ f ,W KDV EHHQ UHFHQWO\ UHSRUWHG WKDW VHUD IURP SDWLHQWV ZLWK DGXOW SHULRGRQWLWLV SRVVHVV KLJK DQWLERG\ OHYHOV WR WKH % JLQJLYDOLV KHPDJJOXWLQLQ ,W LV WKXV VXJJHVWHG WKDW WKH DGKHVLYH VXUIDFH VWUXFWXUHV VXFK DV KHPDJJOXWLQLQ SDUWLFLSDWH LQ % JLQJLYDOLV FRORQL]DWLRQ DQG DQWLJHQLF VWLPXODWLRQ RI WKH KRVW 1DLWR HW DO f +HPDJJOXWLQDWLQJ DFWLYLW\ KDV DOVR EHHQ VKRZQ WR EH D XQLTXH FKDUDFWHULVWLF RI RUDO VWUDLQV RI % DVDFFKDURO\WLFXV SUHVHQWO\ % JLQJLYDOLVf 1RQRUDO VWUDLQV RI % DVDFFKDURO\WLFXV GR QRW KDYH KHPDJJOXWLQDWLQJ DFWLYLW\ 6ORWV DQG *HQFR f &RPSRQHQWV RI EDFWHULD ZKLFK PHGLDWH DWWDFKPHQW WR KRVW WLVVXHV LQFOXGH VXUIDFH VWUXFWXUHV VXFK DV ILPEULDH FDSVXLDU PDWHULDO OLSRSRO\VDFFKDULGH DQG PHPEUDQHDVVRFLDWHG H[WUDFHOOXODU YHVLFOHV 6ORWV DQG *HQFR f )LPEULDO SUHSDUDWLRQV IURP % JLQJLYDOLV KDYH EHHQ IRXQG WR SRVVHVV VWURQJ KHPDJJOXWLQDWLQJ DFWLYLW\ EXW /36 RU SRO\VDFFKDULGH DSSDUHQWO\ GR QRW 2NXGD HW DO f +RZHYHU %R\G DQG 0F%ULGH f KDYH

PAGE 113

UHSRUWHG WKDW DQ RXWHU PHPEUDQH SUHSDUDWLRQ RI % JLQJLYDOLV : PHGLDWHV KHPDJJOXWLQDWLRQ EXW GRHV QRW FRQWDLQ ILPEULDHOLNH VWUXFWXUHV ,Q DGGLWLRQ
PAGE 114

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nKLFK IXQFWLRQ DV KHPDJJOXWLQLQV 7R GHWHUPLQH ZKHWKHU WKHVH FORQHG '1$ IUDJPHQWV DUH DGMDFHQW LQ %DFWHURLGHV FKURPRVRPDO '1$ LH FRQWDLQHG LQ RQH JHQHf RU QRW WKH IROORZLQJ H[SHULPHQWV VKRXOG EH SHUIRUPHG +\EULGL]DWLRQ RI UHVWULFWHG % JLQJLYDOLV FKURPRVRPDO '1$ ZWK '1$ LQVHUWV IURP FORQHV DQG (DFK '1$ LQVHUW VKRXOG K\EULGL]H WR WKH VDPH FKURPRVRPDO '1$ IUDJPHQW LI WKHVH LQVHUWV DUH FRQWLJXRXV RQ WKH FKURPRVRPDO '1$ $SSURSULDWH UHVWULFWLRQ HQ]\PHV VKRXOG EH XVHG WR GLJHVW FKURPRVRPDO '1$ FRPSOHWHO\ LQ RUGHU WR JHQHUDWH IUDJPHQWV Zn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

PAGE 115

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n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

PAGE 116

SURWHLQV ZLOO GHILQLWHO\ FRQILUP WKDW WKH\ DUH WKH VDPH RU GLIIHUHQW SURWHLQV DQG WKXV GHWHUPLQH LI RQH RU PRUH FRPSRQHQWV RI % JLQJLYDOLV PHGLDWH KHPDJJOXWLQDWLRQ ,Q RUGHU WR GHILQH WKH JHQHV ZKLFK HQFRGH WKH KHPDJJOXWLQLQVf VXEFORQLQJ VKRXOG EH SHUIRUPHG 7KH '1$ RI IXQFWLRQDO VXEFORQHV ZLWK WKH VPDOOHVW LQVHUW FRXOG WKHQ EH VHTXHQFHG 7KH SUHGLFWHG DPLQR DFLG VHTXHQFH ZLOO SURYLGH LQIRUPDWLRQ VXFK DV WKH LVRHOHFWULF SRLQW Sf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f ZKLFK DUH VXVSHFWHG WR PHGLDWH DWWDFKPHQW WR KRVW WLVVXHV SURYLGHV D PHDQV WR GHILQH WKH QDWLYH VWUXFWXUHV RI % JLQJLYDOLV UHVSRQVLEOH IRU WKLV DFWLYLW\ 7KH FORQHG KHPDJJOXWLQLQ JHQHV DUH DOVR JRRG FDQGLGDWHV IRU '1$ SUREHV IRU WKH UDSLG LGHQWLILFDWLRQ RI % JLQJLYDOLV LQ FOLQLFDO VDPSOHV 8OWLPDWHO\ WKHVH FORQHG JHQHV PD\ IDFLOLWDWH WKH SURGXFWLRQ RI D YDFFLQH ZKLFK SUHYHQWV WKH FRORQL]DWLRQ RI % JLQJLYDOLV LQ WKH JLQJLYDO VXOFXV DQG SRVVLEO\ WKH SUHYHQWLRQ RI SHULRGRQWDO GLVHDVH

PAGE 117

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f SURWHLQ RI (VFKHULFKLD FROL 3URF 1DWO $FDG 6FL 86$ &KXQJ & + DQG $ / *ROGEHUJ 7KH SURGXFW RI ,RQ FDS5f JHQH LQ (VFKHULFKLD FROL LV WKH $73LQGHSHQGHQW SURWHDVH SURWHLQ /D 3URF 1DWO $FDG 6FL 86$ &LVDU &RDJJUHJDWLRQ UHDFWLRQV EHWZHHQ RUDO EDFWHULD 6WXGLHV RI VSHFLILF FHOOWRFHOO DGKHUHQFH PHGLDWHG E\ PLFURELDO OHFWLQV SS ,Q 5 *HQFR DQG 6 ( 0HUJHQKDJHQ HGVf +RVWSDUDVLWH LQWHUDFWLRQV LQ SHULRGRQWDO GLVHDVH $PHULFDQ 6RFLHW\ IRU 0LFURELRORJ\ :DVKLQJWRQ '& &LVDU 2 ( / %DUVXPLDQ 6 + &XUO $ ( 9DWWHU $ ( 6DQGEHUJ DQG 5 3 6LUDJDQLDQ 'HWHFWLRQ DQG /RFDOL]DWLRQ RI D OHFWLQ RQ 6FWLQRP\FHV YLVFRVXV 7 9 E\ PRQRFORQDO DQWLERGLHV ,PPXQRO

PAGE 118

&LVDU 2 3 ( .ROHQEUDQGHU DQG ) & 0HOLQLWH 6 6SHFLILFLW\ RI FRDJJUHJDWLRQ UHDFWLRQV EHWZHHQ KXPDQ RUDO VWUHSWRFRFFL DQG VWUDLQ RI $FWLQRP\FHV YLVFRVXV RU $FWLQRP\FHV QDHVOXQGLL ,QIHFW ,PPXQ &ODUN : % / / %DPPDQQ DQG 5 *LEERQV &RPSDUDWLYH HVWLPDWHV RI EDFWHULDO DIILQLWLHV DQG DGVRUSWLRQ VLWHV RQ K\GUR[\DSDWLWH VXUIDFHV ,QIHFW ,PPXQ &R\NHQGDOO $ / ) 6 .DF]PDUHN DQG 6ORWV *HQHWLF KHWHURJHQHLW\ LQ %DFWHURLGHV DVDFFKDURO\WLFXV +ROGHUQDQ DQG 0RXUH f )LQHJROG DQG %DUQHV DSSURYHG OLVWV f DQG SURSRVDO RI %DFWHURLGHV JLQJLYDLLV VS QRY DQG %DFWHURLGHV PDFDFDH 6ORWV DQG *HQFRf FRPE QRY ,QW 6\V %DFWHULRO &UDZIRUG $ & 5 6 6 6RFUDQVN\ ( 6PLWK DQG 5 3KLOOLSV 3DWKRJHQLFLW\ WHVWLQJ RI RUDO LVRODWHV LQ JQRWRELRWLF UDWV 'HQW 5HV 6SHFLDO ,VVXH %f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

PAGE 119

*LEERQV 5 DQG (WKHUGHQ &RPSDUDWLYH K\GURSKRELFLWLHV RI RUDO EDFWHULD DQG WKHLU DGKHUHQFH WR VDOLYDU\ SHOOLFOHV ,QIHFW ,UDPXQ f§ *LEERQV 5 DQG 9DQ +RXWH %DFWHULDO DGKHUHQFH DQG WKH IRUPDWLRQ RI GHQWDO SODTXHV SS ,Q ( + %HDFKH\ HGf %DFWHULDO $GKHUHQFH 5HFHSWRUV DQG UHFRJQLWLRQ 6HULHV % 9RO f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n IRUPDWLRQ E\ EDFWHULDO DQWLJHQV LQ SDWLHQWV ZLWK SHULRGRQWDO GLVHDVH $UFK 2UDO %LRO -D\DZDUGHQH $ DQG 0 *ROGQHU 5HDJLQOLNH DFWLYLW\ RI VHUXP LQ KXPDQ SHULRGRQWDO GLVHDVH ,QIHFW ,PPXQ .DJDQ 0 /RFDO ,PPXQLW\ WR %DFWHURLGHV JLQJLYDOLV LQ 3HULRGRQWDO 'LVHDVH 'HQW 5HV '/

PAGE 120

,OO .LOLDQ 0 'HJUDGDWLRQ RI LPPXQRJOREXOLQV $O $ DQG E\ VXVSHFWHG SULQFLSDO SHULRGRQWDO SDWKRJHQV ,QIHFW ,UQPXQ .ODLQIHOGW $ 'HJUDGDWLRQ RI ERYLQH DUWLFXODU FDUWLODJH SURWHRJO\FDQV LQ YLWUR 6HDQG 5KHXPDWRO .ROOHQEUDQGHU 3 ( DQG % / :LOODLPV /DFWRVHUHYHUVLEOH FRDJJUHJDWLRQ EHWZHHQ RUDO DFWLQRP\FHWHV DQG 6WUHSWRFRFFXV VDQJXLV ,QIHFW ,UQPXQ /DHPPOL 8 &OHDYDJH RI VWUXFWXUDO SURWHLQV GXULQJ WKH DVVHPEO\ RI WKH KHDG RI EDFWHULRSKDJH 7 1DWXUH /RQGRQf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f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

PAGE 121

0D\UDQG % & 0F%ULGH 7 (GZDUGV DQG 6 -HQVHQ &KDUDFn WHUL]DWLRQ RI %DFWHURLGHV DVDFFKDURO\WLFXV DQG % PHODQLQRJHQHFXV RUDO LVRODWHV &DQ 0LFURELRO 0F.HH $ 6 $ 6 0F'HUPLG $ %DVNHUYLOOH $ % 'RZVHWW & (OOZRRG DQG 3 0DUVK (IIHFW RI KHPLQ RQ WKH SK\VLRORJ\ DQG YLUXOHQFH RI %DFWHURLGHV JLQJLYDOLV : ,QIHFW ,PPXQ f 0H\HU 7 ) 1 0ODZHU DQG 0 6R 3LOXV H[SUHVVLRQ LQ 1HLVVHULD JRQRUUKRHDH LQYROYHV FKURPRVRPDO UHDUUDQJHPHQW &HOO 0RRQH\ DQG % + :DNVPDQ $FWLYDWLRQ RI QRUPDO UDEELW PDFURSKDJH PRQROD\HUV E\ VXSHUQDWDQWV RI DQWLJHQ VWLPXODWHG O\PSKRF\WHV ,PPXQRO 0RXWRQ & 3 +DPPRQG 6ORWV DQG 5 *HQFR 6HUXP $QWLERGLHV WR 25DO %DFWHURLGHV DVDFFKDURO\WLFXV %DFWHURLGHV JLQJLYDOLVf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f 1DNDPXUD 7 6 )XMLPXUD 1 2EDWD DQG 1
PAGE 122

1LVHQJDUG 5 7KH UROH RI LPPXQRORJ\ LQ SHULRGRQWDO GLVHDVH 3HULRGRQW 2NXGD $
PAGE 123

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f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

PAGE 124

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f 6YHHQ E 5DEELW SRO\PRUSKRQXFOHDU OHXNRF\WH PLJUDWLRQ LQ YLYR LQ UHVSRQVH WR OLSRSRO\VDHFKDULGHV IURP %DFWHURLGHV )XVREDFWHULXP DQG 9HLOORQHOOD $FWD 3DWKRO 0LFURELRO 6FDQG %f 7DND]RH 7 1DNDPXUD DQG 2NXGD &RORQL]DWLRQ RI WKH 6XEJLQJLYDO DUHD E\ %DFWHURLGHV JLQJLYDOLV 'HQW 5HV 7DQQHU $ & 5 & +DIIHU 7 %UDWWKDO 5 $ 9LVFRQWLL DQG 6 6 6RFUDQVN\ $ VWXG\ RI WKH EDFWHULD DVVRFLDWHG ZLWK DGYDQFLQJ SHULRGRQWLWLV LQ PDQ &OLQ 3HULRGRQWDO

PAGE 125

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f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

PAGE 126

:KLWH DQG 0D\UDQG $VVRFLDWLRQ RI RUDO %DFWHURLGHV ZLWK JLQJLYLWLV DQG DGXOW SHULRGRQWLWLV 3HULRGRQW 5HV :LNVWURP 0 % 'DKOHQ DQG $ /LQGH )LEULQRJHULRO\WLF DQG ILEULQRO\WLF DFWLYLW\ LQ RUDO PLFURRUJDQLVPV &OLQ 0LFURELRO
PAGE 127

%,2*5$3+,&$/ 6.(7&+ 6RP\LQJ ZDV ERUQ WR 6DQJXDQ DQG &KDXID -XLMDLWURQJ RQ 'HFHPEHU LQ 7DKPXDULJ .DQFKDQDEXUL 7KDLODQG 6RUQ\LUWJ LV PDUULHG WR 6RUULWKHS 7XUQZDVRUQ DQG KDV VRQV 3DWWDUDZXWK DQG 1DWWDSRO 6KH OLYHG LQ 7DKUQXDULJ XQWLO ZKHQ VKH ZHQW WR 7ULHP ,nGRPVXNVD 6FKRRO LQ %DQJNRN 6KH HQWHUHG .DVHWVDUW 8QLYHUVLW\ LQ -XQH DQG ZDV VXSSRUWHG E\ WKH -RKQ ) .HQQHG\ )RXQGDWLRQ XQWLO VKH UHFHLYHG D %DFKHORU RI 6FLHQFH ZLWK KRQRXUVf LQ IRRG VFLHQFH DQG WHFKQRORJ\ LQ 0D\ 8SRQ JUDGXDWLRQ VKH UHFHLYHG D VFKRODUVKLS IURP WKH 8QLYHUVLW\ 'HYHORSPHQW &RPPLVVLRQ WR SHUVXH D PDVWHUn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

PAGE 128

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 4e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OMWLX+/U\r :LOOLDP % &ODUN 3URIHVVRU RI ,PPXQRORJ\ DQG 0HGLFDO 0LFURELRORJ\

PAGE 129

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ :LOOLDP 3 0F$UWKXU 3URIHVVRU RI ,PPXQRORJ\ DQG 0HGLFDO 0LFURELRORJ\ FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ $QWKRQ\ ) %DUEHW $VVRFLDWH 3URIHVVRU RI 9HWHULQDU\ 0HGLFLQH 7KLV GLVVHUWDWLRQ ZDV VXEPLWWHG WR WKH *UDGXDWH )DFXOW\ RI WKH &ROOHJH RI 0HGLFLQH DQG WR WKH *UDGXDWH 6FKRRO DQG ZDV DFFHSWHG DV SDUWLDO IXOILOOPHQW RI WKH UHTXLUHPHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ $SULO 'HDQ &ROOHJH RI 0HGLFLQH 'HDQ *UDGXDWH 6FKRRO

PAGE 130

81,9(56,7< 2) )/25,'$


UNIVERSITY OF FLORIDA
3 1262 08554 4913